International Journal of Biological Macromolecules 233 (2023) 123407

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

Recombinant protein expression: Challenges in production and folding

related matters
Azadeh Beygmoradi a, Ahmad Homaei a, *, Roohullah Hemmati b, Pedro Fernandes c, d, e
a
Department of Marine Biology, Faculty of Marine Science and Technology, University of Hormozgan, Bandar Abbas, Iran
b
Department of Biology, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran
c
DREAMS and Faculdade de Engenharia, Universidade Lusófona de Humanidades e Tecnologias, Av. Campo Grande 376, 1749-024 Lisboa, Portugal
d
iBB—Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001
Lisboa, Portugal
e
Associate Laboratory i4HB—Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: Protein folding is a biophysical process by which proteins reach a specific three-dimensional structure. The
Folding amino acid sequence of a polypeptide chain contains all the information needed to determine the final three-
Refolding dimensional structure of a protein. When producing a recombinant protein, several problems can occur,
Misfolding
including proteolysis, incorrect folding, formation of inclusion bodies, or protein aggregation, whereby the
Protein aggregation
Post-translational modifications
protein loses its natural structure. To overcome such limitations, several strategies have been developed to
address each specific issue. Identification of proper protein refolding conditions can be challenging, and to tackle
this high throughput screening for different recombinant protein folding conditions can prove a sound solution.
Different approaches have emerged to tackle refolding issues. One particular approach to address folding issues
involves molecular chaperones, highly conserved proteins that contribute to proper folding by shielding folding
proteins from other proteins that could hinder the process. Proper protein folding is one of the main prerequisites
for post-translational modifications. Incorrect folding, if not dealt with, can lead to a buildup of protein mis­
foldings that damage cells and cause widespread abnormalities. Said post-translational modifications, wide­
spread in eukaryotes, are critical for protein structure, function and biological activity. Incorrect post-
translational protein modifications may lead to individual consequences or aggregation of therapeutic pro­
teins. In this review article, we have tried to examine some key aspects of recombinant protein expression.
Accordingly, the relevance of these proteins is highlighted, major problems related to the production of re­
combinant protein and to refolding issues are pinpointed and suggested solutions are presented. An overview of
post-translational modification, their biological significance and methods of identification are also provided.
Overall, the work is expected to illustrate challenges in recombinant protein expression.

1. Introduction folding, proteolysis, or aggregation. Failure of a protein to fold into the

The expression of recombinant protein in the proper host aims at the
production of a protein that has appropriate solubility, folding and ac­
tivity. An important factor in the expression of recombinant protein
solutions is the composition and optimization of the environment. Pro­
tein folding is a biophysical process in which polypeptides assume a
specific three-dimensional (3D) structure that endows them with given
biological functions [1–3]. When a cell is induced to synthesize a given
protein, several problems are likely to surface, including incorrect
correct 3D structure typically results in the production of inactive pro­
teins, but it may also prompt modified and/or toxic functionalities.
Moreover, to have their natural active state, which requires a stable,
specific and biologically functional 3D structure, proteins unfold and
refold constantly throughout their active lives [4–7]. Many diseases are
triggered by the incorrect folding of proteins, thus they are often called
conformational diseases [8–10]. Protein misfolding may thus cause the
onset of diseases such as phenylketonuria [11], Parkinson’s disease
[12], trauma in amyotrophic lateral sclerosis [13], cystic fibrosis [11],

* Corresponding author at: Department of Marine Biology, Faculty of Marine Science and Technology, University of Hormozgan, Bandar Abbas, P.O. Box 3995,
Iran.
E-mail address: a.homaei@hormozgan.ac.ir (A. Homaei).

https://doi.org/10.1016/j.ijbiomac.2023.123407
Received 26 October 2022; Received in revised form 13 January 2023; Accepted 20 January 2023
Available online 25 January 2023
0141-8130/© 2023 Elsevier B.V. All rights reserved.
A. Beygmoradi et al.
familial neurohypophyseal diabetes insipidus [14], spastic paraplegia
[11], Creutzfeldt-Jakob disease [15], scrapie [16] and carbonic anhy­
drase II deficiency syndrome [17]. These problems could be prevented
through the optimization of recombinant protein production and
folding. On the other hand, the expression of recombinant proteins and
post-translational modifications are relevant for addressing the biolog­
ical functions of genes and to perform biochemical, enzymatic and 3D
protein structure studies [18,19]. To date, the knowledge gained on
post-translational protein modifications has contributed to significantly
improve diagnostic and therapeutic strategies for a variety of metabolic
disorders, including diseases such as neurological diseases, diabetes, and
cardiovascular diseases [20,21]. Post-translational modifications of
proteins have attracted a great deal of attention because of their role in
the structural and functional changes of proteins, as they affect many
essential biological processes [22]. Applications of recombinant proteins
include diverse fields, such as medicine, agriculture and enzyme in­
dustry, hence many studies have been done on recombinant proteins and
the associated production of bioactive compounds [23–30]. The opti­
mization of recombinant proteins expression relies on various parame­
ters such as expression vector, culture medium composition, growth
temperature and expression chaperones [24,31,32]. By modifying the
initial protein sequence, protein accumulation can be effectively pre­
vented, but this method is rarely used due to the unpredictable conse­
quences of structural changes [33]. An alternative approach to produce
recombinant protein is de novo protein design, which led to the pro­
duction of recombinant proteins with specific functions and activities
that are not observed in nature [34].
Typically, the production of recombinant protein targets high pro­
tein rate synthesis, high host cell density and, most importantly, high
product quality [35]. Low-cost protein production is an important goal
in the development of pharmacotherapy and bio-industrial processes
[36]. Thus, only large-scale processes can cope with the growing de­
mand, and those processes require cost-effective approaches [37]. So
far, recombinant therapeutic proteins such as insulin, human growth
hormone and antihemophilic factor VIII have been produced using as
host cells either prokaryotes (e.g., Escherichia coli) or eukaryotes (e.g.,
Saccharomyces cerevisiae cells, insect, mammalian, human and plant
cells) [31,32,38–40]. E. coli cells were the first (and simplest) host to
produce recombinant therapeutic proteins in the 1980s. Among
eukaryotic expression systems, yeast has many advantages such as
relatively low-cost culture media, rapid cell growth, absence of endo­
toxins and a diversity of post-translational changes mechanisms that
include, e.g., folding and glycosylation [41,42]. Also, yeasts have the
potential to produce industrially recombinant proteins due to their
powerful promoters that control gene expression. However, despite the
potential of microbial cells as hosts, mammalian cells have gained the
upper hand as primary host cells in recent years [43,44].
Inclusion bodies, protein rich aggregates that accumulate in the
cytosol, nucleus or periplasm of cells [45], are often formed due to
overexpression of genes in foreign hosts, creating the need for further
treatments such as unfolding and refolding [34]. The formation of in­
clusion bodies is thus a major hurdle in the industrial production of
functional recombinant proteins. Genetic engineering has been shown to
provide a way out of this limitation, hence enabling mass production of
the proteins envisaged. Therefore, most biopharmaceutical products are
currently produced by recombinant methods [27]. Plenty of post-
translational modifications of the polypeptide chain occur simulta­
neously or after protein folding, which involves proteolytic cleavage and
the addition of functional groups to the protein. Gaining insight on such
modifications is important in the biosynthesis of pharmaceutical prod­
ucts, biological processes and in the treatment of diseases such as cancer,
diabetes and heart diseases where more than one gene is involved, since
in most cases the production of a recombinant protein as a drug requires
some post-translational modifications of the protein. So, any researcher
who starts a new project immediately must work out the proper strategy
to produce a protein that has high activity and quality. The ability to
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International Journal of Biological Macromolecules 233 (2023) 123407
express, activate and purify recombinant proteins in large quantities
that can meet human needs is an important issue in medical biotech­
nology, industrial processes and in the production of valuable com­
mercial products. Protein folding has important applications in
biotechnology, including protein engineering and de novo protein
design with new functions. Therefore, molecular targeting of post-
translational modifications of proteins is a promising strategy for drug
development, cancer treatment, as well as other inflammatory diseases,
given their role and potential. Table 1 shows the biological importance
of these modifications.
This review highlights the relevance of recombinant proteins, briefly
addresses production issues and identifies key factors that impact the
structure, function and activity of said proteins, with particular focus on
folding issues. As deeply related to these, post-translational modifica­
tions are also abridged, namely their nature, biological significance, and
methods of identification, not forgetting a brief insight on their role in
metabolic disorders and disease.
2. Folding problems during the production of recombinant
proteins and examining the working-out of those problems
Mass production of high-quality recombinant protein is an important
part of industrial processes. The main goal in the production of recom­
binant proteins is high protein rate synthesis, high host cell density and
most importantly high product quality [35]. Theoretically, the process
of obtaining recombinant proteins is very easy, yet many problems may
occur along the way, such as inclusion bodies, protein aggregation,
protein inactivity, incorrect folding, and incorrect modification of the
protein after translation and even lack of protein production. The na­
ture, structure, and function of proteins is determined by the coding
gene. Accordingly, the first step in identifying the true cause of an ab­
normality and understanding the protein structure is genome
sequencing [34]. Optimization of protein folding conditions is an
important part in the production of recombinant proteins, which is
mainly achieved through experimental observations and laboratory
tests. To expedite the latter, high throughput screening methods coupled
to dedicated analytical tools have been implemented [89–92] and da­
tabases have been created, e.g., https://www.re3data.org/repository/
r3d100010553 (assessed on June 24, 2022), which contribute to more
comprehensive screening efforts. Correct folding of a polypeptide chain
follows first-order kinetics while the process of protein aggregation in
insoluble form is a quadratic kinetic function in competition with correct
folding [93]. In addition, in the formation of inclusion bodies, the for­
mation of insoluble primary masses called nuclei of mass bodies plays a
key role. Therefore, the lower the production of recombinant proteins,
the lower the probability of forming insoluble and inactive forms [94].
The expression kinetics of the recombinant protein depends on the
expression temperature and on the inducer concentration. Lowering the
temperature slows down the production of recombinant protein and
provides enough time for the protein to fold [95]. In this case, reducing
the concentration of the intermediate of the folding path reduces the
possibility of the formation of primary nuclei of the mass body. How­
ever, lowering the temperature also has its problems. Thus, the optimum
temperature for some chaperones is around 30 to 37 ◦ C [96] and their
activity decreases sharply at temperatures under 18 ◦ C. Therefore, in­
cubation at temperatures under 20 ◦ C for expression requires the use of
complementary systems. In laboratory tests, protein folding is often
more efficient with low protein concentrations and low temperatures,
and protein production at lower temperatures has a profound effect on
protein quality [97,98]. However, protein folding occurs in human cells
at 37 ◦ C (or above, during fever), with very high protein concentrations
[99]. In addition to temperature, inducer concentration is another
effective factor in expression. Decreasing the concentration of the
inducer reduces the expression of a protein at the transcriptional level
and plays a role in reducing the median concentration in the folding
pathway, ultimately reducing the likelihood of inclusion body
A. Beygmoradi et al.
Table 1
Introduction of post-translational modifications and their biological importance.
Type of Biological importance
modification
Acetylation Histone acetylation: Effect on cellular homeostasis, protein
stability, regulation of various transcription factors, molecular
chaperones and cellular metabolism [46,47]
Glutamic acid acetylation: Conversion of glutamic acid to N-
acetylglutamic acid, an important intermediate step for
ornithine synthesis in bacteria [48]
Acetylation of skeletal proteins: Increased microtubule
acetylation suppresses SIRT2 and impaired mitochondrial
function causing chronic progressive external
ophthalmoplegia syndrome [49]
Hypersensitivity to lysine β-amyloid peptide leads to cognitive
impairment, age-related memory impairment and Alzheimer’s
disease [47]
Carbonylation In response to oxidative stress, large amounts of by-products
are formed, including nitrotyrosine and protein carbonyl,
which lead to autoimmune diseases such as hypocitrullinemia,
systemic sclerosis and fasciitis, mitochondrial disorders, high
oxidative stress, and glutathione loss [44,50]
Irreversible carbonylation of the protein to methionine sulfone
as an indicator of oxidative stress and Alzheimer’s disease as
well as other neurodegenerative diseases [51]
Glycosylation O-glycosylation: has an important role in Alzheimer’s disease
by reducing the formation of neurofibrillary tangles in nerve
cells [52]
Leads to various diseases including cancer, liver cirrhosis,
diabetes and exacerbation of HIV infection [53,54]
Prion glycosylation: A cell surface protein and a transmissible
factor that determines the final outcome of disease in the host
[55]
Glycosylation of apolipoprotein E: Role in Alzheimer’s disease,
atherosclerosis and immune responses [56]
Inadequate or incomplete Fc receptor glycosylation for
immunoglobulin A: Effect on IgA-mediated immune response
and affecting many diseases including HIV, alcoholic liver
cirrhosis and other neuropathies [54,57]
Hydroxylation Proline hydroxylation: Activation of antioxidant defense
against hypoxia through hypoxia inducible factor (HIF) [58]
Asparagine and proline hydroxylation: Acts as a “hypoxic
switch” to induce HIF under hypoxic conditions [59]
Phenylalanine hydroxylase: Deficiency in phenylalanine
hydroxylase (an enzyme that converts phenylalanine to
tyrosine) causes diseases such as phenylketonuria and
hyperphenylalaninemia [60]
Tyrosine hydroxylase: As a molecular target for the treatment
of hypertension [60,61]
Tryptophan hydroxylation: An important regulatory step in
the production of serotonin, an important neurotransmitter
[62]
Methylation Histone methylation: Along with histone acetylation, plays a
role in controlling cellular RNA synthesis, activation and
inhibition of specific RNAs and cell metabolism [63,64]
Histone methyltransferases: Regulation of gene expression by
targeting lysine residues in histones [63]
Lysine methylation: The main role in aggregation and storage
of mitochondrial ATP synthase as aggregate in lysosomal
bodies [60]
Methionine methylation: Excessive accumulation of
homocysteine (a by-product of this modification) plays a major
role in cardiovascular disease as well as in neurological
disorders such as Parkinson’s disease, although an optimal
amount of homocysteine is necessary for normal body function
[65]
Nitration Nitrotyrosine (as a by-product of this modification) plays an
important role in regulating various pathways of cellular
signals and is associated with many neurodegenerative
diseases, lung diseases, inflammation, cardiovascular disease
and cancer [66,67]
Another harmful product of this modification, which consists
of NO and superoxide ions, together with nitrotyrosine in the
development of amyloid β-peptide toxicity and in Alzheimer’s
disease [68]
Cellular nitroproteins are associated with the inflammation of
3
International Journal of Biological Macromolecules 233 (2023) 123407
Table 1 (continued )
Type of Biological importance
modification
the human intestinal epithelium, a symptom of irritable bowel
syndrome [69]
Palmitoylation Role in regulating and modulating G protein-associated cell
signaling pathways [70]
Role in neuroinflammatory response including demyelinating
diseases as well as T cell autoimmune responses [71]
Dysfunctional palmitoylation of γ-secretase, a component of
β-amyloid, in Alzheimer’s disease negatively affects the
functional potential of neurons [72]
Lack of palmitoylation of cysteine in Huntington’s protein
causes Huntington’s disease to increase neurotoxicity and
formation of inclusion bodies [73]
Phosphorylation Effect on mitogen-activated protein kinase signaling with
tyrosine kinase receptor pathway and cell cycle-related
proteins such as cyclin-dependent kinases, regulation of
different signaling pathways, control of cell growth and
differentiation, immune response, apoptosis and maintenance
of cellular homeostasis with proper balance between protein
kinases and phosphatases [74–76]
Irregular phosphorylation plays a role in neurological diseases
such as Parkinson’s disease, dementia and Lewy body
accumulation [77]
Histidine phosphorylation role in regulating metabolic
signaling pathways in eukaryotic cells [78]
Serine/threonine and tyrosine phosphorylation role in cancer
progression and cell differentiation and proliferation [79,80]
Sulfation Sulfated proteins: An important role in protein/protein
interactions, G protein receptor signaling, chemokine
signaling and immune responses [81]
Tyrosine sulfation: Role in cellular calcium transport,
induction of protein-10 by γ-interferon [82], autoimmune
response, HIV infection, lung disease, multiple sclerosis, cell
enzyme regulation [83] and induction of chemokine signaling
in diseases Pulmonary, such as chronic obstructive pulmonary
disease and asthma, leading to exacerbation of airway
inflammation [84]
Ubiquitination The target protein is determined by the two complexes
anaphase-promoting complex and Skp1-Cullin-F-box protein
complex for proteasome degradation [85].
Role in modulating various cellular functions such as cell
proliferation and differentiation, gene transcription,
autophagy, apoptosis, immune response, DNA repair, nerve
damage, myogenesis and inflammatory and stress responses
[86]
Impact on many life-threatening diseases such as cancer,
neurodegenerative disorders, HIV infection, herpes and liver
diseases [87,88]
formation. It has been reported that reducing transcription to an
inducible level of 1 % can be effective in the active production of re­
combinant protein [18]. Finally, the expression time of a protein plays a
key role in its successful production. In general, as the production time
decreases, the probability of aggregation of the half-folded in­
termediates decreases, resulting in the formation and growth of the
nuclei of the mass body. Simultaneous expression of genes encoding
expression chaperones and target proteins has also been shown to
deliver the soluble form of the protein [100]. Increasing the concen­
tration of chaperones or lowering the temperature facilitates the process
of protein folding [101]. In general, protein folding proceeds thermo­
dynamically to produce a stable end-product. Proteins that are highly
unstable, even in the presence of chaperones, are likely to misfold [102].
Therefore, failure of polypeptides, production of signal domains from
multi-subunit protein complexes, lack of formation of disulfide bonds
normally involved in protein structure, or lack of post-translational
modifications, including glycosylation, make thermodynamic stability
impossible. In addition, it is clear that different types of chaperones act
in unison. Therefore, overproduction of a single chaperone will be
ineffective. For example, high production of DnaK alone causes plasmid
instability, which decreases with concomitant production of DnaJ
[103]. Table 2 lists some of the problems during the production of
A. Beygmoradi et al. International Journal of Biological Macromolecules 233 (2023) 123407

Table 2
Some problems related to the production of recombinant protein and suggested solutions.
Problems Side effects Suggested solutions References

Protein aggregation Impact on protein morphology Binding of proline by folding intermediates [33,104,105]
Primary protein sequence change Decreased protein concentration
Defective deletion of signal peptides Activation of cells at the time of induction
Temperature stress, pH Protein misfolding Optimization of expression conditions including [97,98,104,106]
and induction Abnormal conformation temperature, gene promoter power, gene expression
Protein aggregation induction time and inducer concentration
Lowering the temperature reduces the rate of transcription,
translation and refolding, thus favoring correct folding.
Formation of incorrect Protein misfolding Simultaneous expression of Dsb family proteins (with [33,97,98,107,108]
disulfide bond Increased physical accumulation of protein disulfide bonds)
Accumulation of inclusion bodies Increased pH
Inactive protein production
Formation of inclusion Overexpression of heterologous proteins Use the appropriate expression environment [106,109–112]
bodies Decreased protein activity Simultaneous overexpression of molecular chaperones
Insoluble protein production without Changes in growth conditions, such as growth temperature
biological activity (preferably decreasing temperature), inducer concentration,
and induction time
Reduce solubility Formation of inclusion bodies Low temperature expressiona [113–115]
Increase solubility by placing a tag on the N-terminal or C-
terminal of the protein
Use of solubility enhancing proteins such as maltose binding
protein (MBP) and NusA
Use of solubility enhancing molecular chaperones such as
prolyl cis-isomerases, thioredoxin and dsbA
Decreased efficiency and Low activity in the peptidase signal Addition of small molecules or proteolytic groups to the [116–118]
quality of expression culture medium
Methionine removal from N-terminus by methionine amino
peptidaseb
Protein misfolding Decreases in the stability, solubility and Increased expression of various chaperones [97,98,100,104,106,110,115,119,120]
activity of recombinant proteins Prevent the formation of insoluble and non-native folding
intermediaries
Use the appropriate expressive environment
Expression at low temperature increases solubility and folds
correctlyc
Increased cell density Decreased production of recombinant protein Proper nutrition solutions [121–123]
due to nutrient restriction and high cell
concentrations
Contamination in very high cell
concentrations
Toxicity of recombinant Unnecessary or lethal action of the Synthesis control by promoters [124–127]
protein recombinant protein on the host cell Dealing with toxic effects using a variety of plasmids with
Decreased production of recombinant protein the encoding gene of lysozyme T7
a
This is ascribed to the hydrophobic interactions that determine the secretory bodies are temperature dependent.
b
This only occurs in proteins whose second amino acid is Ala, Gly, Ser, Thr, Pro or Val.
c
This is ascribed to the hydrophobic interactions that determine the secretory bodies are temperature dependent. On the other hand, lowering the temperature
probably slows down the transcription, translation and refolding rates and thus favors the correct folding.

recombinant proteins, adverse effects, and solutions to these problems.
Another issue during the production of recombinant protein is
refolding. Refolding implies the shift in protein conformation from
unfolded to folded and depends on the denaturant concentration
[11,12]. It is accepted that the refolding process in vitro is a suitable
model for understanding the mechanisms by which the polypeptide
chain reaches its natural conformation in the cellular environment. In
fact, many denatured proteins, even those with disrupted disulfide
bridges, are able to refold [15]. Refolding of proteins that have been
denatured by a variety of factors, including the addition of denaturants a
pH and temperature shift, has been observed. Refolding has been ach­
ieved through pH adjustment, denaturant removal and addition of ad­
ditives such as denaturants (at low concentrations), stabilizers, e.g.,
glycerol and aggregation inhibitors [28,128]. When protein concentra­
tions are low, renaturation requires the use of large volumes of refolding
buffer [28,129]. Protein refolding consists of two steps. In the first step,
the denatured protein is diluted with a diluent buffer containing deter­
gent, which prevents protein aggregation. In the second step, the
detergent/protein solution is diluted with a buffer containing cyclo­
dextrins. The second step is effective in correcting and refolding the
synthesized protein [28,129]. Protein destabilizers usually accelerate
protein aggregation. During refolding, the reduction of denaturant
concentrations to low titers and the stabilization of the target protein
structure inhibits protein aggregation [33,105,116]. Some amino acids,
including arginine and some of its derivatives, are classified as protein
aggregation inhibitors due to their moderate binding to the protein.
Accordingly, arginine, arginine hydrochloride, arginine amide and
glycine amide have been increasingly used as additives in refolding
buffers, leading to increased refolding efficiency as aggregation is
minimized [28,129–133]. Moreover, several chromatographic methods
including gel filtration, hydrophobic interaction chromatography and
ion exchange, have been used to facilitate the refolding step. Table 3
highlights key factors that should be considered when optimization of
the refolding buffer is envisaged.
3. Other methods for solving problems related to folding
Hybrid protein technology provides an alternate approach to address
protein folding issues, since it has been shown that it increases the sol­
ubility of the studied protein [138–140]. Common hybrid proteins
include maltose-binding protein (MBP), glutathione S-transferase, thi­
oredoxin, and NusA.MBP contribute to increase the solubility of the final
product due to folding and acts as a general chaperone, thus preventing
the aggregation of semi-folded intermediates in the folding process

4
A. Beygmoradi et al. International Journal of Biological Macromolecules 233 (2023) 123407

Table 3 using proline, glycine and arginine [153,154]. The addition of metal
Factors affecting the optimization of the refolding buffer. ions as cofactors to the medium has been reported to increase the pro­
Factors Mechanisms References duction of several recombinant active proteins. In addition, metal ions
facilitate the formation of the final structure of the recombinant protein
pH Refolding observed within pH 5 to 9. [130,134,135]
Experimentally, refolding should be through interaction with semi-folded intermediate structures. Finally,
performed at least one unit higher or lower cofactors stabilize the folded proteins [155]. To maintain the proper
than the isoelectric pH of the protein under efficiency of protein folding and to protect against the harmful conse­
study. quences of environmental changes, several other strategies have been
Temperature Effect on refolding kinetics through increased [136]
formation of active structure
developed, including the correct folding of recombinant proteins by
Perform many refolding processes, single molecular chaperones and protein quality control systems, which are
temperature, at temperatures of 4 ◦ C or close discussed below.
to room temperature
Salt The salt concentrations used in many [137]
4. The role of molecular chaperones in proper folding and
concentrations refolding processes are around 50 to 100
mM. prevention of protein aggregate
Regenerative The presence of reducing agents such as 1,4- [134,135]
factors dithiothreitol (DTT) and β-mercaptoethanol Molecular chaperones protect proteins during the folding process to
in the refolding buffer prevents the formation prevent misfolding and are involved in folding, refolding, quality and
of unwanted disulfide bonds.
Use of metal ions Stabilization of folded proteins [130,134,135]
structure of proteins, preventing the accumulation and ability of natural
Successful refolding proteins [6]. Choosing the right chaperone can play an important role in
Add chemical Different concentrations: L-arginine M [130–133] increasing the stability or proper folding of recombinant proteins. The
foldases 0.4–0.5, glycerol 10–50 %, SDS 0.1 %, folding process is protected by molecular chaperones and directs them to
sucrose/glucose 0.4 M, Tris 0.4–1 M and
their natural structure [156]. Molecular chaperones, GroES and GroEL
EDTA 20 mM
chaperones play an important role in preventing improper folding, the
opening of erroneously folded polypeptides and subsequent protein
[141,142]. accumulation, and even enable the detection of unfolded or aggregated
Thioredoxin is highly soluble and its expression in the expression proteins. As a result, they facilitate protein folding [6,156–158]. Chap­
system E. coli led to the production of 40 % of total cellular proteins in a erones have ATPase activity and perform their function specifically. In
completely soluble form [143]. Thioredoxin can also be present at both E. coli, chaperone facilitates the folding of bound polypeptides. Heat
ends of the recombinant protein [144], since both amine and carboxyl shock proteins are classified into several families based on the molecular
ends are free. This, along with the small size of thioredoxin (about 12 weight of their monomers including Hsp40, Hsp60, Hsp70, Hsp90 and
kDa), can reduce the problems associated with the spatial inhibition of Hsp100 [159–161]. Hsp70 constitutes the major part of chaperone
the attached protein. Thioredoxin has also been shown to contribute to proteins in all organisms and acts rapidly under heat shock [160]. Hsp70
proper folding of proteins containing disulfide bonds [97]. is a suitable drug target and binds to protein chains and hydrophobic
Compared to other hybrid proteins, the role of NusA is less well regions of the folded protein, thus preventing their improper aggrega­
known. However, the solubility of this protein is higher when compared tion, while Hsp60 binds folding mediators [161,162]. Therefore, Hsp70
to other hybrid proteins. is a practical and useful strategy for cancer treatment [159,163]. Mo­
Despite the efficiency of hybrid protein technology, a number of lecular chaperones are divided into three categories based on the
problems have been reported associated with this approach, including mechanism of action and type of interaction with substrate proteins,
the production of soluble aggregates, lack of biological activity, and including folding chaperons, holding chaperons and disaggregate
unpredictability of the solubility of a protein in the presence of a hybrid chaperons [164]. Folding chaperons mediate ATP-dependent alteration
protein [97]. of their substrates by refolding or non-folding, thereby preventing the
Foldases can also be used to address improper folding [145]. These formation of inclusion bodies by reducing aggregates and proteolyzing
enzymes are divided into two general categories, namely periplasmic proteins that have been misplaced. These chaperones include DnaK and
foldases and cytoplasmic foldases, depending on the compatibility with GroEL. Holding chaperons such as IbpA and IbpB can co-operate with
the type of expression system selected. Periplasmic foldases are effective folding chaperons to keep some of the folded proteins on their surface
only next to the secretory expression systems and outside the cytoplasm and under certain conditions, and to protect heat-denatured proteins
and do not have a direct effect on the cytoplasm like oxidizing agents from irreversible aggregates [165]. Disaggregate chaperones such as
[146]. Cytoplasmic foldases can enter the cytoplasm. Therefore, they clpB and HtpG that inhibit aggregation and stimulate the solubility of
can be used in secretory expression systems or in intracellular expression accumulated stress-inducing proteins [166].
systems. β-Casein is a relatively low molecular weight protein (~24 kDa) that
Another factor in the production of active recombinant proteins is exhibits chaperone-like activity in vitro. β-Casein solubilizes proteins
the addition of ethanol to the culture medium. The presence of ethanol previously aggregated and prevents aggregation by attenuating hydro­
in the culture medium has been reported to triple the solubility of some phobic intramolecular interactions between unfolded or incompletely
proteins. This increase in solubility appears to occur through stimulation folded intermediates that are responsible for the aggregation process
of a number of cell chaperones, especially Dank and GroEL [147]. It has [167]. Glycerol, a naturally occurring polyol, has been shown to in­
also been reported that the use of polyols (such as sorbitol and glycerol) crease the kinetics of the folding process, while preventing the aggre­
increases the solubility of recombinant proteins by competing with gation of proteins, thus playing a role in protein stability [168,169].
water molecules to form hydrogen bonds with the major protein chain
[148,149]. 5. Protein quality control systems
Sugars, amino acids and metal ions have also been reported to play a
role in the proper folding of several proteins [150,151]. Sucrose is the If the expression of the recombinant gene is low, factors such as
most well-known sugar with folding activity [152]. This disaccharide mRNA stability, the occurrence of secondary structures at the 5′ mRNA
increases the solubility of recombinant protein by inducing the forma­ end, rare codons, and the weak Shine Dalgarno sequence may reduce
tion of hydrogen bonds in the main protein chain and indirectly by expression [163]. The first and fastest cell reaction is the removal of
inducing Dnak and Ibp chaperones at the transcriptional level. incorrectly folded proteins [170]. Protein quality control systems have
Regarding the effect of amino acids, most studies have been performed evolved to protect cells against both unwanted products during

5
A. Beygmoradi et al.
translation and damaged proteins. Protein quality control systems act by
controlling folding and counteracting aggregation by binding to
damaged polypeptide chains to eliminate misfolded monomers and
incorrectly folded proteins that result of the accumulation of incorrectly
folded proteins. Moreover, increased titers of incorrectly folded proteins
may hamper the protein degradation system and ultimately result in the
destruction of the entire cell (Fig. 1). These systems also include a
number of additional side factors and intracellular proteases that regu­
late the activity of molecular chaperones and proteases [156]. The
functions of the protein quality control systems include: the production
and maintenance of functional proteins; the removal of defective ribo­
somal products; and the elimination of damaged and abnormal proteins
[171]. The endoplasmic reticulum lumen contains high concentrations
of chaperones and folding enzymes that play an important role in con­
trolling protein quality [172]. When the immune system fails to main­
tain healthy cells, a phenomenon called apoptosis or necrosis occurs,
leading to cell death [173].
6. An overview of post-translational modification of proteins
One of the final steps in protein biosynthesis is the post-translational
modification of proteins. During this extensive process, proteins
Fig. 1. Protein quality control system. If protein folding is not done properly and misfolded proteins are formed, the ubiquitin proteinase system (UPS) can degrade
misfolded proteins from the endoplasmic reticulum (ER) by Endoplasmic reticulum-associated protein degradation (ERAD). If the protein quality control system fails
to do its job properly, the cells may become apoptotic, leading to cell death.
6
International Journal of Biological Macromolecules 233 (2023) 123407
undergo a series of chemically reversible or irreversible changes after
translation. Post-translational modifications of proteins play an impor­
tant role in the production of several functional proteins from a single
gene and in the structure, function and regulation of many proteins
[174,175]. There are several types of modifications, including phos­
phorylation, glycosylation, acetylation, ubiquitylation, sumoylation,
methylation, palmitoylation, myristoylation, prenylation, hydroxyl­
ation, GPI anchoring, ADP-ribosylation, citrullination, S-nitrosylation,
oxidation and sulfation (Fig. 2). Further details on post-translational
modifications vocabulary and updates on this field, see https://www.
uniprot.org/help/post-translational_modification (assessed January 7,
2023), a database curated by the UniProt consortium [176]. Post-
translational modifications play an important role in various cell func­
tions, e.g., phosphorylation in intracellular signaling, ubiquitination in
regulating protein stability, histone acetylation and methylation in
transcription regulation, and glycosylation in cell surface signaling.
Glycosylation is one of the most common and complex forms of
protein modification in eukaryotic cells. Glycosylation plays an impor­
tant role in the biological activity, folding, stability and solubility of
proteins [177–179]. Glycoproteins are a significant part of therapeutic
proteins [180] and it has been shown that glycosylation enhances their
therapeutic properties by increasing their half-life, antibody-mediated
A. Beygmoradi et al.
Fig. 2. Different types of post-translational modifications.
cytotoxicity, and immunogenicity [181], alongside with stability and
proper folding [182,183]. The biological role of protein glycosylation
can be divided into three categories, related to protein stabilization,
modulation of glycoprotein properties, and identification of glycan-
binding proteins [184,185]. Folding and subsequent processes are
associated with N-glycan bonds, hence blocked N-glycosylation can lead
to protein disorders such as partial loss of folding and dysfunction [186].
Glycosyl transferase UGGT1 is the main enzyme in controlling the
quality of folding in protein [187]. Both O- and N-glycosylation have
been reported in yeasts [188,189]. Among the various strains of yeast,
Saccharomyces cerevisiae has been traditionally used as host for recom­
binant protein production, however, Pichia pastoris has been shown to
present a more successful expression system [190,191]. Bacterial
glycosylation in the human pathogenic system Campylobacter jejuni is
encoded by a gene locus called pgI (protein glycosylation) that can be
functionally transmitted to the E. coli expression host [192–194]. This
gene locus encodes a glycosylation pathway linked to asparagine resi­
dues in the target protein that is functionally similar to the pathway of
eukaryotes and archaea [195]. The easiest way to produce glycosylated
proteins is through protein expression in E. coli [98,159]. Using the pgI
N-glycosylation system in C. jejuni, several products, including glyco­
conjugated vaccines have been synthesized [98,196,197]. This bacte­
rium has glycosylated predisposing cells including
oligosaccharyltransferases and lipid-linked oligosaccharides, which
activate glycosylation sites in target proteins for more efficient glyco­
sylation [198]. In yeasts, N-glycosylation increases protein expression in
cells and during the protein folding process, and plays an important role
in the production and secretion of heterologous proteins that require N-
glycosylation for proper folding [199]. Therefore, deletion of N-glyco­
sylation sites in yeast cells inhibits the expression of recombinant
7
International Journal of Biological Macromolecules 233 (2023) 123407
proteins [189]. Up-to-date, a large number of bacterial glycosidases that
play an important role in bacterial pathogenesis, nutrient uptake,
display increased stability and effective function and modulate immune
response have been identified [200]. In addition, proteomic engineering
and metabolic analysis have been used to significantly improve the ef­
ficiency of protein glycosylation in E. coli [201].
Acetylation is one of the major post-translational modifications
involved in the regulation and normal function of many cellular pro­
cesses including chromatin stability, cell cycle control, cell metabolism,
transcription, gene regulation, cellular homeostasis, protein stability
and enzymatic activity [202–205]. In eukaryotes, most proteins are
terminally acetylated in the N-terminal region [202,206]. This process
affects various aspects of the function of eukaryotic proteins expressed
in bacteria [207]. In general, acetylation is catalyzed by two enzymes,
lysine acetyltransferase and histone acetyltransferase, and ultimately
lead to the transfer of the acetyl group from acetyl coenzyme A to the
epsilon amine group of lysine. This protein change, which is conserved
across phylogeny [208], is rarely seen in bacteria and, unlike eukary­
otes, occurs after translation [209]. Most of the knowledge gathered so
far on protein acetylation involves eukaryotes; still, in the last decade
research dedicated to gain insight on the role of acetylation in bacteria
has steadily increased, as this mechanism is likely to impact how bac­
teria dynamically regulate several metabolic functions [208]. Mulvihill
et al. have shown that co-expression of a member of the Nα-acetyl­
transferase complex of yeast with its target substrate proteins in E. coli
can successfully produce a number of acetylated proteins of human and
yeast origin [98,210]. These findings suggest that a wide range of re­
combinant acetylated proteins could potentially be produced in E. coli,
as exemplified in recent works [211].
Phosphorylation is one of the most important reversible
A. Beygmoradi et al.
modifications after translation of protein in the cytosol or cell nucleus in
eukaryotes, which involves the addition of a phosphate group to an
amino acid and leads to changes in protein activity and cellular meta­
bolism [212]. Phosphorylation plays an important role in regulating
protein function, message pathways in biological systems, maintaining
cellular homeostasis, transcription, cell motility, regulating cell growth,
cell survival, cellular metabolism, and apoptosis [212–215]. This
modification is performed by protein kinases that catalyze the transfer of
phosphate from ATP to serine, threonine, or tyrosine. E. coli has been
widely used as host for the expression of phosphorylation in many re­
combinant proteins, including: human protein phosphatase-2Cα [216]
and glutathione S-transferase-nucleoside diphosphate kinase (GST-
NDK) [217]; several response regulators from Pseudomonas stutzeri
RCH2 [218]; and cardiac myosin binding protein C [215], among others
[215].
Methylation is one of the most important and reversible processes in
post-translational modification of proteins. So far, this process has been
mostly studied in histone proteins, although this pattern has been
changing lately, as methylation of non-histone protein lysine has been
recently acknowledged as a dominant modification occurring in several
proteins [219]. DNA methylation enables additional information to be
transferred to DNA, and this epigenetic information can change the
timing and targeting of cellular events [220]. DNA methylation occurs at
the C-5 or N-4 cytosine position and at the N-6 adenine position and is
catalyzed by enzymes known as DNA methyltransferases (MTases)
[220,221]. In mammals, 5′ units of cytosine are methylated in CpG
dinucleotide sequences [222]. This process is performed by a group of
methyl group-transporting enzymes or DNA methyltransferases using S-
adenosylmethionine as methyl donor, which is responsible for methyl­
ating cytosine bases [223]. This compound has suitable methyl groups
for methylation of e.g., RNA, histones, neurotransmitters, membrane
phospholipids and various proteins [224].The transfer of methyl groups
from S-adenosylmethionine to histones is mediated by histone methyl­
transferase, which results in the suppression or activation of the
expression of various genes [225]. In prokaryotes, DNA methylation
occurs in both cytosine and adenine units, and is also involved in pro­
cesses such as regulation of gene expression, repair, control of cell
proliferation, and defense against foreign DNA. In eukaryotes, methyl­
ation in coordination with other epigenetic changes, regulates gene
expression and chromatin structure [226]. Specific methyltransferases,
such as DNA cytosine MTase, responsible for methylating the C-5 cyto­
sine position in the CC(A/T)GG sequence, DNA adenine methylase
(Dam), responsible for methylating the N-6 adenine methylation in the
GATC sequence, cell-cycle regulated methylase responsible for methyl­
ating the N-6 adenine of GAnTC have been shown not be associated with
cognate restriction enzyme [220,227]. Preliminary studies have shown
that Dam regulates the expression of some genes in E. coli in the 5′ -
GATC-3′ sequence [227], a pattern that seems to be confirmed [228].
Additionally, it has been suggested that Dam, besides being accountable
for GATC methylation may also act as a transcriptional repressor, in a
methylation-and GATC-independent manner [229]. Dam has also been
tentatively related to the development of antibiotic resistance, namely
when acrAD-containing efflux pumps are involved [230], moreover,
Dam has been shown to regulate virulence gene expression in some
E. coli strains [231].
S-nitrosylation of proteins is a reversible post-translational modifi­
cation that consists of covalent bonding of a nitric oxide (-NO) group to
thiol cysteine within a protein. This modification regulates enzyme ac­
tivity, protein interactions, protein function control, and signal trans­
duction pathways [232,233]. S-nitrosylation regulatory roles across
numerous physiological processes can be found in bacteria, yeasts,
plants and mammals [234,235]. This process transfers much of the
pervasive effect of nitric oxide on cellular signal transduction and pro­
vides a mechanism for redox-based physiological regulation [233] as
recently reviewed [236]. Nitration begins when amino acids are exposed
to nitrates, genotoxic, or oxidative stress [237]. One of the by-products
8
International Journal of Biological Macromolecules 233 (2023) 123407
of nitration is nitrotyrosine, which plays an important role in the cell,
including regulation of several cellular signaling pathways, gene regu­
lation, and induction of an antioxidant defense response [67,238].
Sulfation is a post-translational modification involving a sulfo group
(− SO3H) or its anion form and hydroxy or amine moieties of various
substrates that is observed in a wide diversity of molecules many mol­
ecules including proteins, carbohydrates, and lipids [239]. It has been
observed in both proteins and glycoproteins, namely through tyrosine
sulfation [240] and is also observed in glycans, mainly in hexoses and N-
acetyl hexosamines [241,242]. Sulfation is involved in various biolog­
ical processes including detoxification, intracellular transport of
secreted proteins, hormone regulation, molecular identification, cellular
signaling, interactions between proteins, homeostasis, visual function,
and virus entry into the cell [241]. Biological sulfation is involved in the
synthesis of sulfonated glycosaminoglycans such as heparin, creatine
sulfate, chondroitin sulfate, dermatan sulfate, and heparan sulfate. This
modification is commonly reported for secretory and membrane pro­
teins [243,244]. Tyrosine sulfation, first discovered in bovine fibrin­
ogen, is one of the major post-translational changes [245] and has been
found to occur in animals and plants [240]. To investigate post-
translational tyrosine sulfation modification, three tyrosine-sulfated
hemostatic proteins including fibrinogen, factor V, and heparin
cofactor II, and three proteins that had not yet been tyrosine-sulfated but
had potential, including protein S, prekallikrein, and plasminogen, were
evaluated. Methods for identifying this modification included immu­
nosuppression and SDS-PAGE gel electrophoresis. After electrophoresis,
all six proteins were sulfated. The radioactive bands of the three sulfated
proteins were subjected to autoradiography. Excess radioactive stains
confirmed the sulfate binding of these three proteins. It has previously
been shown that plasminogen with O-glycosylation and N-glycosylation,
prekallikrein with N-glycosylation and S protein with γ-carboxylation
and β-hydroxylation were post-translationally modified [246].
Palmitoylation or protein S-acylation is a reversible modification
that covalently converts fatty acids such as palmitic acid to cysteine (S-
palmitoylation) and serine and threonine (O-palmitoylation) [247].
Palmitoylation adds lipid groups to the protein structure, thus it in­
creases the hydrophobicity of proteins and eases their docking to
membranes. Palmitoylation plays an important role in cell trafficking,
membrane stimuli, cell signaling, cancer, neuronal cell transport, and
modulation of protein-protein interactions [248,249]. The first samples
of enzymes to mediate palmitoylation, for example acyl transferases,
have only recently been identified in yeast [250]. Most Ras proteins are
modified by a palmitoyl lipid moiety after translation through a thio­
ester bond, but the mechanism of this is unclear [250]. Nerve proteins
and synapses are modified by S-palmitoylation. Thus, impaired S-pal­
mitoylation causes changes in synaptic strength and ultimately leads to
neurological disorders including Alzheimer’s and Huntington’s diseases
[72,73,251].
Myristoylation is a common co-translational lipid modification
observed in many organisms, including animals, plants, fungi, protozoa,
and viruses, in which a myristoyl group, derived from myristic acid, is
covalently linked by an amide bond to the alpha-amino group of glycine
N-terminal residue of several proteins [252–254]. This modification can
be added as a simultaneous or post-translation. N-myristoyltransferase
catalyzes the covalent addition of myristic acid to the N-terminal glycine
of a protein in the cytoplasm of cells. Myristoylation plays an important
role in membrane targeting, cell proliferation, differentiation, cell cycle
and regulation of cell signaling pathways, and apoptosis [254–256].
Myristoylation and palmitoylation are two interconnected modifica­
tions. Myristoylation can produce transient membrane interactions that
enable proteins to anchor membranes but are easily separated [255].
Palmitoylation further allows the anchor to be harder and separate from
the membrane. These two modifications are important for G protein
receptor pathways [243,255].
Sumoylation is another type of post-translational modification of a
protein in which members of a small ubiquitin-related modifier family of
A. Beygmoradi et al.
proteins, such as Sumo (Small ubiquitin-like modifier), bind reversibly
to the substrate [257]. Sumoylation is involved in important biological
processes including transcription control, chromatin organization, and
DNA repair. Disruption of this process leads to irreversible disorders
including Parkinson’s, Alzheimer’s, brain failure, cancer, diabetes and
cardiovascular disease.
ADP-ribosylation is a reversible post-translational modification that
involves the addition of one or more units of ADP-ribose to a protein
[258,259]. This modification is involved in many cellular processes,
including cellular signaling, cell differentiation, DNA repair, gene
regulation, stress, and apoptosis [260–262]. Disorders of ADP-
ribosylation in some cases lead to abnormalities such as cancer and
neurological disorders [263–265].
Citrullination is another post-translational modification whereby an
arginine residue is converted to citrulline. This increases the hydro­
phobicity of the protein and causes changes in the protein folding, thus
affecting its structure and function. Conversion of arginine to citrulline
can have important consequences for the structure and function of
proteins, because arginine is positively charged at neutral pH, while
citrulline is uncharged [266]. Histones are particularly sensitive to the
changes in electrostatic charge [267]. This protein modification is
associated with apoptosis and cell differentiation [268]. Protein citrul­
line modification was first discovered in autoimmune diseases such as
rheumatoid arthritis [268–270]. Citrulline proteins are potential targets
for the diagnosis or treatment of cancer. Modifications associated with
citrullination disrupt the stability of proteins and thus damage DNA
[267]. Carbamylation is the chemical modification of lysine to homo­
cytroline, while citrulline is the enzymatic conversion of arginine to
citrulline [271]. Due to structural similarities, these two amino acids are
difficult to distinguish.
Hydroxylation is a common oxidative process and one of the most
important detoxification reactions in cells and is further facilitated by a
group of enzymes called hydroxylase. Hydroxyl derivatives are prom­
ising in nature for many drugs and treatments of metabolic disorders
[272]. Proline hydroxylation has significant effects on cell structural
physiology. Prolyl 4-hydroxylase is a α2β2 tetramer that catalyzes the
pos-translational hydroxylation of prolines into collagen. Its recombi­
nant production is targeted, since prolyl 4-hydroxylase is one of the
promising candidates for medical and biotechnological applications,
including cosmetics and pharmaceutical industries [273–275].
Carbonylation is an irreversible post-translational modification that
can result from excessive oxidative stress in the cell, leading to the
formation of inactive proteins and the development of many diseases,
including autoimmune diseases and cancer [276]. Carbonylation can be a
valuable modification for the identification of oxidative stress and ni­
trogen (ROS and RNS), mitochondrial diseases, neurological disorders,
and cardiovascular disease [277].
Ubiquitination is a multi-enzymatic post-translational modification
that ultimately leads to the covalent attachment of a small regulatory
protein, ubiquitin, typically to lysine residues of the protein. The
modification targets proteins for degradation and recycling. Protein
degradation occurs by labeling the amino group of the side chain of a
lysine residue of the targeted protein with ubiquitin, a small protein
ubiquitous in eukaryotic cells [278].
Table 4 lists some modifications that occur during the production of
recombinant protein.
7. Methods of diagnosis and identification of post-translational
modifications
Identification of post-translational modifications of proteins and
their related sites is performed by various experimental and computa­
tional methods. In computational methods, post-translational modifi­
cations are determined using experimental data analysis. Experimental
methods of identification somewhat vary, depending on the type of
modification. In vitro ubiquitination technique is a good method to
9
International Journal of Biological Macromolecules 233 (2023) 123407
study the degradation of proteins [309,310]. PCR can be used to
quantify the presence of the desired modification in a specific location.
Mass spectrometric methods, occasionally involving an HPLC step, can
be used to detect glycosylation and glycan analysis. The glucose groups
are first released, either enzymatically or chemically as glycoproteins,
then the released oligosaccharides are examined by LC/MS [311].
Isolation of fluorophores or chromophores added to the glycan reducing
end is performed by electrophoresis. To better detect glycosylated pro­
teins, reagents that have a high affinity for glycans (e.g., glycans and
lectin-binding antibodies) are used [312]. To obtain information about
glycosylated proteins, endoproteinase is first degraded and then glyco­
peptides are analyzed by LC/MS or HILIC-MS/MS methods. This method
allows to identify the location of the modified proteins or the glycosyl­
ation status of proteins carrying a specific glycan fraction [313]. The
standard method for establishing protein palmitoylation is derived from
the labeling of [3H] metabolic palmitic acid. This method requires a
large amount of radioactive input tags to detect palmitoylation. Table 5
lists some methods for identifying protein modifications based on the
role and method of operation.
MudPIT method, a chromatography-based proteomic approach, was
used to identify palmitoylated proteins in yeast. Based on this technique,
protein palmitoylation in S. cerevisiae was evaluated and confirmed
[331]. Moreover, S-palmitoylation can be detected using biochemical
methods including radioactive alkylation reaction and metabolic label­
ing in which synthetic fatty acid analogues are used [332]. Citrulline can
be stained with paired dyes such as rhodamine or biotin and then
detected by ELISA or mass spectrometry [333,334]. Using this tech­
nology, more antigens can be found with citrulline modifications that
provide new targets for the diagnosis and treatment of cancers [267].
Kenner and co-workers confirmed the phosphorylation of human eIF2α
protein based on the combined phos-tag and SDS-PAGE techniques
[335]. Meisrimler and co-workers used a combined phos-tag method
with two-dimensional electrophoresis to evaluate phosphorylation-
dependent changes in enzyme activity [336]. Kitchen and co-workers
used the phos-tag method to analyze AQP4 phosphorylation [337].
The identification was based on modified myristoylated proteins, MS,
nano LC-MS/MS and FPLC methods. Based on this method, the myr­
istoylated proteins of factor 1 ADP - ketone-modified ribosylation with
hydrazine were labeled with fluorophore and then identified by fluo­
rescence imaging. This method is an effective model for describing lipid-
modified proteins [338]. The ADP ribosylation process is visible by
HPLC, LC-MS. The basis for identifying carbamylation and citrulline
modifications involves ELISA and Western blot [271].
8. Conclusion
Recombinant proteins have a wide array of applications that bio­
industries, health and pharma. To cope with the growing demand, the
production of recombinant proteins has increased dramatically in
various areas of the industry. Such trend has further stressed the need to
adequately identify and properly address several issues that emerge
during recombinant protein production, such as level of expression ag­
gregation, misfolding or proper refolding, to name a few. Proteins that
have been aggregated may exhibit unwanted properties such as reduced
or inactive biological activity or other side effects. Protein misfolding
can cause irreversible abnormalities, such as disease in organisms or
significant industrial problems in the production of recombinant pro­
teins. Therefore, different strategies have been suggested to cope with
each problem, such as the selection of optimal operational conditions,
reduction of production times, use of chaperones or proper refolding
buffer. To evaluate the efficiency of those strategies, quality control
systems have been proposed. Since the production of valuable recom­
binant proteins and therapeutic applications requires post-translational
modifications, these were overviewed, their biological significance
identified and methods for their identification highlighted. So far, bac­
terial strains of E. coli, the yeasts of P. pastoris and S. cerevisiae have
 A. Beygmoradi et al.
Table 4
Expression of some modified proteins in eukaryotic and prokaryotic expression systems.
Host Modification Recombinant enzyme or protein Extracted from Expression vector Purification method MWa (Da) Analytical methods Other characteristics References

S. cerevisiae O-glycosylation β-Mannase Trichoderma reesei pAJ401 Chromatography A – 53 IEF analysis C-Terminal C rich in Ser-Thr- [279]
Sepharose CL-4B Pro Enzyme displays
similarities to the cellulose
binding domains of fungal
cellulase
E. coli O-glycosylation N-acetylgalactosaminyltransferase Plesiomonas pEt 23a(+) Ni-NTA affinity 40 Analysis by LC – ESI Expression in the bacterial [180]
SHuffleT7 2 shigelloides chromatography – MS method cytoplasm simultaneously
with UDP-GlcNAc
Conversion of UDP-GlcNAc
to UDP-GalNAc by 4-
epimerase
P. pastoris N-glycosylation Cold-adapted serine protease Glaciozyma pPIC9 99 – Maximum activity at pH 7.0 [280]
antarctica strain and temperature 20 ◦ C
PI12
P. pastoris N-glycosylation Elastase – – – – – Stimulation of expression by [189]
adding more glycosylated N-
glycans sequence (Asn-Xaa-
Thr sequence) to appropriate
locations
The Asn-Xaa-Thr sequence is
more glycosylated than the
Asn-Xaa-Ser
P. pastoris N-glycosylation Elastase – pPICpK – – – Replacement of Thrby Ser at [189]
strain N-glycosylation sites (N36,
KM71 N43, N212, N264, and N280)
10

Increased N-glycosylation by
switching at N36, N43,
N212, and N280 (conversion
at N212 and N280 resulted in
increased expression and
decreased in N36 and N43)
Stimulatory or inhibitory
effects on glycoprotein
expression by replacing Thr

International Journal of Biological Macromolecules 233 (2023) 123407

with Ser
P. pastoris N-glycosylation Elastase – – – 33 (non- Conversion of Asn residue to [281]
glycosylated) Gln at three N-glycosylation
35 (nono- sites N212, N43 and N280 by
glycosylated) mutation
37 (bi- Mutations proved
glycosylated) detrimental to protein
expression leading to
decreased expression
P. pastoris N-glycosylation Elastase Pseudomonas pPIC9 K MW similar to N-glycosylation in N51 or [282]
KM71 aeruginosa [281] N93 positions increases and
in N11 or N127 positions
decreases glycosylation level
P. pastoris N-glycosylation Ovalbumin pGAPZαAandpRS426 DEAE-Sepharose™ Western blotting Mutation at N292 and N311 [283]
X-33 FF chromatography analysis and conversion ofAsn to Gln
reduces expression
compared to the wild type
N-glycan at N292 position
mandatory for proper
secretion and folding
(continued on next page)
 A. Beygmoradi et al.
Table 4 (continued )
Host Modification Recombinant enzyme or protein Extracted from Expression vector Purification method MWa (Da) Analytical methods Other characteristics References

Improper folding of
ovalbumin prevents
glycosylation of Asn-292,
which leads to loss of protein
secretion
S. cerevisiae N-glycosylation Wild-type enzyme phospholipase B Cryptococcus pYES2: 75 (Wild type: Analysis by Western Conversion of Asn to Ala via [284]
(PLB1) and glycosyl phosphatidyl neoformans CnPLB1andpYES2: non- blotting with an mutagenesis at N-
inositol without anchor version CnPLB1GPI− glycosylated) anti-Express glycosylation sites at
(PLB1GPI− ) 120 antibody positions N56, N430 and
(glycosylated) N550 ease protein expression
S. cerevisiae N-glycosylation α-Amylase Schwanniomyces YEp13 DEAE-cellulose (DE- 59.4 (wild Expression at two N- [285]
occidentalis SWA2 52) column type) glycosylation sites including
chromatography 57.4 (mutant Asn-134 and Asn-229
type) Replacement of Asn by Ala or
Gly alone or in combination
Replacement in N229
reduces expression and in
N134 had no effect on
expression
Replacement by Ala or Gly
results in decreased MW of
the protein by 2 KDa
S. cerevisiae N-glycosylation Chain A – ricin Increased expression as Asn [286]
replaced Gln at two N-
glycosylation sites in N10
and N236
E. coliCLM24 N-glycosylation AcrA and IFN-α2b pACYC 23.7 (IFN-α2b) Western blotting Insertion of Glu and Asn at [287]
11

analysis N102 and N104,

respectively, with the pelB
signal sequence and a hexa-
his tag sequence at C
-terminal. Led to significant
increase in protein
glycosylation efficiency with
glucose uptake and

International Journal of Biological Macromolecules 233 (2023) 123407

phosphorylation in the path
of the carbon preservation
agent
Increased glycosylation
efficiency of AcrA and IFN-
α2b by 1.69-fold and 2.2-
fold, respectively
E. coli N-glycosylation Eukaryotic N-glycoproteins C. jejuni pACYC184 Hydroxylapatite 41,011 Da ESI-MS analysis Expression of glycosylated [26]
chromatography proteins with the GlcNAc-
Asn sequence
Modification of bacterial
glycans using trans
glycosylation in vitro
E. coli N-glycosylation AcrA protein with pgl locus from C. jejuni Glycosylated AcrA The expression loci of pgl [288]
C. jejuni analysis by SDS- and AcrA contained a signal-
PAGE and dependent peptide
immunoblotting, AcrA protein with double
MS glycosylation at N123 and
N273
Glycosylated folded proteins
(continued on next page)
 A. Beygmoradi et al.
Table 4 (continued )
Host Modification Recombinant enzyme or protein Extracted from Expression vector Purification method MWa (Da) Analytical methods Other characteristics References

(AcrA) in vitro
Location of glycosylation
sites in the flexible parts of
folded proteins
E. coli N-glycosylation Secretory glycoprotein with locus C. jejuni pTrc99A Ni-NTA affinity Western blot Transfer of cell-expressed [289]
BL21(DE3) pgl from C. jejuni chromatography analysis and proteins to the glycosylated
blotting with anti- locus of pACYCpgl or
His (top) or hR6P pACYCpglmut
(bottom) antibody
E. coli N-glycosylation Trimannosyl chitobiose glycans – – – – Production of recombinant [290]
protein and transfer of
glycans to specific Asn
residues in target proteins by
an artificial pathway in
E. coli
Glycan biosynthesis by 4
eukaryotic
glycosyltransferases
including uridine
diphosphate-N-
acetylglucosamine
transferases Alg13 and Alg14
and mannosyltransferases
Alg1 and Alg2
Transfer of eukaryotic
proteins using the PglB locus
from C. jejuni
12

E. coli N-glycosylation AcrA gene C. jejuni strain pBR322, pEC415 Ni2+ affinity 17 NanoLC-MS/MS Isolation of non-glycosylated [192]
Top 10 81–176 chromatography analysis proteins by SBA agarose
resin
E. coli CLM24 N-glycosylation Novel cell-free glycoprotein Ni-NTA affinity – Western blot and Glycoprotein synthesis by [291]
chromatography nanoLC – MS/MS the pgl locus glycosylation
analysis system in the C. jejuni
genome
Novel cell-free glycoprotein

International Journal of Biological Macromolecules 233 (2023) 123407

substrate
Ability to use different OSTs
and LLOs
E. coli Phosphorylation Catalytic subunit of cAMP- – pT7-7 and pLWS-3 Mono-S ion – Identification of [292]
BL21 (DE3) dependent protein kinase exchange phosphorylation locations at
chromatography S10, S139, S338 and T197
positions by (32Pi)-
orthophosphate
Autophosphorylation for
modification of catalytic
subunits during expression
Heterologous protein kinases
responsible for in vivo
phosphorylation
E. coli Phosphorylation Phospholipase A2 – pRSET Ni2+-chelate 110 – Stimulation of cell growth [293]
chromatography factor by increasing
phosphorylation of
phospholipase A2 on serine
residues
Protein kinase C and
(continued on next page)
 A. Beygmoradi et al.
Table 4 (continued )
Host Modification Recombinant enzyme or protein Extracted from Expression vector Purification method MWa (Da) Analytical methods Other characteristics References

microtubule-dependent
protein p42 kinase have
different patterns of
phosphorylation of
phospholipase A2
Phosphorylation of
microtubule-dependent
protein kinase p42
Phospholipase A2 domain
contains a microtubule-
dependent protein kinase
consensus sequence
Expression of protein kinase
C at phosphorylated sites in
all three enzyme domains in
combination
Mediation of the combined
function of these kinases and
high effect on growth factor
stimulation on arachidonic
acid release through
phospholipase A2 activation
E. coli Phosphorylation EnvZ and OmpR, expression – Gel filtration 37 Western blotting Gamma-(32P)-ATP [294]
regulators of ompF and ompC analysis phosphorylation in vitro
expression
E. coli Phosphorylation Osteopontin Mouse pGEX-3× Glutathione-agarose 26 (Pure fusion – Transfer of phosphate group [295]
affinity protein with to OmpR transcription
13

chromatography GST producing activating protein

factor)
60
(recombinant)
E. coli Phosphorylation CAMP-dependent protein kinase Mouse pT7–7 Mono-S ion 9.6 and 14 – A fully phosphorylated [296]
BL21 (DE3) exchange recombinant enzyme
chromatography containing four phosphates
E. coli Phosphorylation Protein A-MuLV (mouse leukemia Mouse 62 Bacteria expressing A-MuLV [297]

International Journal of Biological Macromolecules 233 (2023) 123407

– – –
virus genome) protein contain new
phosphorylated, phosphate-
related proteins in tyrosine
residues
P. pastoris Hydroxylation Tetramer 4-hydroxylase prolyl and Chondrosia pPIC/ColCH 240 (αP4H) Nano-LC/Q-TOF MS Ability to hydroxylate the [275]
a non-fibrillar procollagen reniformis (marine 59 (βP4H) and MS/MS analysis natural substrate of prolyl 4-
polypeptide sponge) 58.7 (βP4H) hydroxylase at both the X
68 (βP4H-am). and Y positions in the Xaa-
Yaa-Gly collagen sequence
E. coli Hydroxylation Human phenylalanine hydroxylase – PET and pMAL Optional 51 Optimal pH about 7. [298]
BL21 chromatography Recombinant proteins
containing amylose endured degradation by host
resin (purification of cell proteases
host cell proteases)
Hydroxyl apatite or
Protein-Pak DEAE
HR column
chromatography
(recombinant
hPAH)
(continued on next page)
 A. Beygmoradi et al.
Table 4 (continued )
Host Modification Recombinant enzyme or protein Extracted from Expression vector Purification method MWa (Da) Analytical methods Other characteristics References

E. coli O-methylation O-methyltransferase 2 Streptomyces – 38 TLC, HPLC, LC-MS, O-methyltransferase [299]

avermitilis MA-4680 LC and NMR methylation with flavonoids
Expressed recombinant
enzyme with His-tag
marking the transfer of
methyl to the group of 7-hy­
droxyl metabolites
E. coli Methylation Protein arginine methyltransferase Chromatography 100 (GST- Methylation Simultaneous expression of [300]
7 of human glutathione- PRMT7) analysis by TLC and human glutathione S-
Sepharose 4B beads safety purification transferase-human arginine
FLAG-PRMT7 methyltransferase 7 (GST-
PRMT7) protein for
methylation
Methylation of this protein in
sequences of H4 histones,
myelin base protein, a
fragment of human
fibrillarin, and spliceosomal
protein SmB
As the substrate
concentration increased, the
symmetric dimethylation
decreased compared to the
monomethylation
E. coli Methylation DNA methyltransferase 3a Mouse pET28a Ni-NTA affinity 140 Recombinant protein of a [301]
chromatography new methyltransferase
catalyzes the transfer of
14

methyl groups to non-

methylated substrates with
similar performance to semi-
methylated substrates
Methylation activity peaked
at pH 7 and 30 mM KCl
E. coli Methylation The cglIM gene encodes the Corynebacterium – – – Significant similarity of [302]
enzyme 5-cytosine glutamicum ATCC amino acid sequences with 5-

International Journal of Biological Macromolecules 233 (2023) 123407

methyltransferase 13032 methyl cytosine residues
producing methyltransferase
enzymes
E. coli Methylation Gene encoding methionine Hyperthermophilic JM1Q9 DEAE-SepharoseCL- The enzyme is the [303]
aminopeptidase archaea Pyrococcus 6B chromatography evolutionary boundary
furiosus between bacteria and
eukaryotes.
E. coli Acetylation Acetyl CoA synthase gene Schizochytrium sp. – – – Acetyl CoA synthase gene [304]
TIO1101 expression, to reduce the
negative effects of acetate
accumulation and damage
on cell growth
Excessive expression in
Schizochytrium sp. TIO1101
increased pH and decreased
the acetate concentration
and production of by-
products
E. coli Acetylation Acetyl CoA synthetase gene JM109 Anion-exchange 47 (α: AcdAI) – N-terminal amino acid [305]
BL21(DE3) chromatography 25 (β: AcdBI) sequence encoding of both
(continued on next page)
 A. Beygmoradi et al.
Table 4 (continued )
Host Modification Recombinant enzyme or protein Extracted from Expression vector Purification method MWa (Da) Analytical methods Other characteristics References

Archeo subunits to acdAI and acdBI

hyperthermophilic genes
Pyrococcus furiosus The recombinant hetero-
tetramer enzyme had
molecular and catalytic
properties similar to acetyl
coenzyme A synthetase
purified from P. furiosus
E. coli Acetylation Parvalbumin (A calcium-binding E. coli Ni-NTA agarose 16.610 Da – Metal-free parvalbumin [207]
BL21(DE3) protein in vertebrates) N-Terminal affinity (RimI) exposed to Nt-acetylation
Acetyltransferases (RimI, RimJ, chromatography 22.688 Da and inhibition of binding of
and RimL) TCA protein (RimJ) this modification by Ca2+
precipitation 20.681 Da Structural stabilization of
method, Sephadex (RimL) parvalbumin induced by
G-25 gel-filtration Ca2+ prevents Nt-acetylation
method, Ni-NTA Activation of RimI and RimJ
agarose affinity in vitro relative to
chromatography parvalbumin with N-
terminal Ser or Ala
E. coli Nitrilation Nitrile hydratase (NHase) Rhodococcus sp. N- pET23c Purification by 47 – NHase had coding genes [306]
T7 771 ammonium sulfate including NHase regulator 2,
expression precipitation and NHase regulator 1, amidase,
system butyl-Toyopearl NHase α subunit, NHase β
column subunit and NHase activator
A modifier of a cysteine
ligand (αCysll2) of
recombinant nitrile hydrate
15

was translocated to a
cysteine-sulfonic acid similar
to native nitrile hydrate.
Reversible inactivation of
recombinant nitrile hydrate
by nitric oxide
E. coli S-nitrosylation Class III alcohol dehydrogenase Branchiostoma Talon metal affinity 140 and 110 – Enzyme protected against [232]
called S-nitrosoglutathione floridae resin nitrosative stress

International Journal of Biological Macromolecules 233 (2023) 123407

reductase chromatography Twice as much activity of
recombinant class III alcohol
dehydrogenase purified as S-
nitrosoglutathione reductase
than formaldehyde
dehydrogenase
E. coli Oxidation Protein disulfide isomerase Conus snail pET28a-sPDI Ni2+-chelating 56.5 (protein LC-MS Successful expression of [307]
affinity disulfide peptidyl-propyl-cis-trans-
chromatography isomerase) isomerase and lt14a
1393.55 and conotoxin in a highly soluble
1393.56 (lt14a form by fusion with Conus
isomers) disulfide isomerase protein
Conus disulfide isomerase
protein catalyzed the
oxidative process and acted
as fusion partner in the
expression of recombinant
conotoxin
E. coli Oxidation Vanadium dodecamer-dependent Corallina officinalis – – 768 (12 × 64 Successful refolding [308]
bromo peroxidase (Seagrass) kDa) conditions created lead to
(continued on next page)
 A. Beygmoradi et al.
Table 4 (continued )
Host Modification Recombinant enzyme or protein Extracted from Expression vector Purification method MWa (Da) Analytical methods Other characteristics References

with increased bromoperoxidase

Quaternary function
structure Optimal temperature of
refolded protein 80 ◦ C, pH
range between 5.5 and 10
and increased stability in
organic solvents
Yeast Palmitoylation ERF2 and ERF4 genes encoded by S. cerevisiae pFLAG-MAC GSH-agarose affinity 41 (Erf2p) Immunoblotting Simultaneous purification [250]
Ras protein acyltransferase chromatography 26 (Erf4p) analysis with anti- and expression of Erf2p (with
GST antibody (top a cysteine-rich domain) and
panel) or anti-FLAG Erf4p
antibody (bottom Ras protein acyltransferase
panel) required the Erf2p/Erf4p
complex for activity
Mutations in Erf2p-protected
16

Cys 203, Cys 189 and His 201

residues inhibit Ras protein
acyltransferase activity
Erf2p role in palmitate
transport
ERF2 and ERF4 are the first
palmitoyl transferase genes
identified to encode a Ras

International Journal of Biological Macromolecules 233 (2023) 123407

GTPase

IEF: isoelectric focusing; LC-ESI-MS: Liquid Chromatography-Electrospray Ionization-Mass Spectrometry; QTOF-MS: Quadrupole Time of Flight Mass Spectrometer.
a
MW: molecular weight.
A. Beygmoradi et al.
Table 5
Introduction of detected methods of modified proteins.
Type of technique Performance
Mass spectrometry Detect most post-translational modifications
Amino acid sequencing of peptides
Determination of molecular weight of peptides and
proteins
Radiometry Evaluation of the role of specific kinases
Evaluation of phosphorylation status
Detection of molecular weight of phosphorylated
protein
Phos-tag View different phosphorylated and non-
phosphorylated proteins
Facilitate the analysis of Western blot samples
TUBEs (Tandem Ubiquitin Binding Applied method for purification of
Entity) polyubiquitinated proteins from cell extracts
Protects ubiquitinated proteins against degradation
and ubiquitination
CHIP (chromatin Purification of chromatin to produce short DNA
immunoprecipitation) fragments Location identification and cystic
synthesis of histone in the promoter
Identify areas in the genome where histones have
specific modifications
SUBEs (SUMO Binding Entities) Identification of tumor suppressor pathways in
human cells
PRISM (Protease-Reliant Confirmation and location identification of
Identification of SUMO SUMOylated proteins
Modification)
MudPIT (Multi-Dimensional Identification of palmitoyl proteins
Protein Identification Powerful technique in the analysis of highly complex
Technology) protein samples
PULSE-CHASE labeling Detection of half-lives of various S-palmitoylation
proteins
Identify palmitoylation sites
PalmPISC Ability to accurately determine S-palmitoylation
proteins Determine the exact locations of S-
palmitoylation modifications
Analysis of palmitoylation in tumor cell lines
BST (Biotin Switch Technique) Locate the modified proteins
His-Tag Switch Identification of modified peptides
Identification of S-nitrosylated modification sites in
proteins
Direct detection by ESI-QTOF mass Identify the location of modifications
spectrometry
Gold Nanoparticles (AuNPs) Identification of modifications after translation of
proteins
Simultaneous isolation and enrichment of thiol-
containing or modified peptides
served as suitable expression host for most modifications including N-
glycosylation, but it may be foreseen that with on-going developments
other microbial hosts may emerge. Moreover, post-translational modi­
fications have proved a major mechanism for regulating various protein
functions and affecting many biological processes and cellular function,
yet further dedicated research is on-going to gain further insight on the
mechanisms underlying such modifications. It can be foreseen that in a
near future such development will prove useful in the implementation of
rational approaches to more efficient recombinant protein production
but also in the design of drugs and therapies to assist in the heteroge­
neous diseases such as cancer and neurological diseases, among other
acute chronic diseases.
Declaration of competing interest
The authors declare that there is no conflict of interest regarding the
publication of this paper.
17
International Journal of Biological Macromolecules 233 (2023) 123407
Method References
Sometimes with HPLC, LC/MS or HILIC-MS/MS [311,313]
Using P-gamma-ATP 32 [314,315]
Using a selective phosphate-binding molecule to isolate phosphorylated [316–318]
proteins in acrylamide and agarose gels
[319–321]
Using antibodies and immunoprecipitation against the desired histone [322,323]
modification
[324]
Chemical blockade of all free lysines by analysis of SUMO-specific [325]
proteases and detection of unblocked lysines based on MS technique
A method based on MS [326]
Exposure of cells labeled with radioactive label for a short time [327]
[327]
The basis of the method of blocking free thiols with thiosulfonate, [328,329]
digestion of proteins with trypsin and incubation of peptides obtained
with avidin or its derivatives, washing of peptides, blocking of free
thiols by NEM
Purification of His-Tag-containing proteins by nickel column [330]
chromatography and one-dimensional electrophoresis, trypsin gel
digestion.
Identification of the modified peptide by LC-MS/MS
This method is limited to isolated proteins or peptides [330]
The basis of this method is the reduction of cysteine residues first by [330]
alkylated iodoacetamide and then proteolysis of the protein.
Following this digestion, incubation of peptides with AuNPs and
reaction with S-nitrosylated cysteine residues, elution of nanoparticles
using DTT and investigation of MS modification sites
Acknowledgements
The authors express their gratitude to the research council of the
University of Hormozgan for financial support during the course of this
project.
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