Received: 8 February 2021 | Revised: 20 June 2021 | Accepted: 27 August 2021

DOI: 10.1002/bit.27935

REVIEW

Sortase A: A chemoenzymatic approach for the labeling of cell

surfaces

Poonam Kumari1 | Sujoy Bowmik1 | Sudipto Kumar Paul1 | Bidisha Biswas1 |

1 1
Sanjay K. Banerjee | Upadhyayula Surayanarayana Murty |
Velayutham Ravichandiran2 | Utpal Mohan2

1
Department of Biotechnology, National
Institute of Pharmaceutical Education & Abstract
Research (NIPER), Guwahati, Assam, India
Sortase A, a transpeptidase enzyme is present in many Gram‐positive bacteria and
2
Department of Medicinal Chemistry, National
Institute of Pharmaceutical Education &
helps in the recruitment of the cell surface proteins. Over the last two decades,
Research (NIPER), Kolkata, West Bengal, India Sortase A has become an attractive tool for performing in vivo and in vitro ligations.
Sortase A‐mediated ligation has continuously been used for its specificity, robust-
Correspondence
Utpal Mohan, Department of Medicinal ness, and highly efficient nature. These properties make it a popular choice among
Chemistry, National Institute of protein engineers as well as researchers from different fields. In this review, we give
Pharmaceutical Education & Research
(NIPER), Kolkata, WB 700054, India. an overview of Sortase A‐mediated ligation of various molecules on the cell surfaces,
Email: mohan.utpal@gmail.com which can have diverse applications in interdisciplinary fields.

KEYWORDS
enzyme, ligation, S. aureus, Sortase A

1 | INTRODUCTION chemically modifying the tyrosine, aspartate, or glutamate amino

Cell surface modification is one of the powerful tools in bioengi-
neering to conjugate tag/functional molecules on the cell surface
(Fox & Sarkar, 2014). These modifications are essential to carry out
biophysical and structural studies. Protein labeling with synthetic
molecules opens up a new approach to study the specific function of
the proteins in the living system, cel–cell interaction as well as the
interaction of cells with extracellular environment (Abbina et al.,
2017; George et al., 2004). Cell surface modification is quite a chal-
lenging task due to the presence of side‐chain groups present in the
surface proteins. The prerequisites of cell surface labeling are the
specificity and versatility, which aids in manipulating cell surfaces.
Wide array of methods are available for the cell surface labeling
such as chemical‐based, genetic‐based, and enzyme‐based (Sahoo,
2012; Sijbrandij et al., 2013; Wu et al., 2009). Chemical‐based
methods are the oldest and most used, exploited both covalent as
well as noncovalent approaches. Most common methods to func-
tionalize proteins utilize either lysine or cysteine amino acid reside
with thiol or amine derivates such as N‐hydroxysuccinimidyl ester or
maleimides. In addition, other methods have been developed by
acids (Hermanson, 2013). With time, other approaches are also been
discovered, to attain specificity, greater yield, sensitivity, and reaction
rate (Tiefenbrunn & Dawson, 2010). All these methods have their
own limitations. Genetic‐based methods are highly selective and ef-
fective. The genetic‐based fluorescent labeling is widely used for the
different molecules like proteins, peptide, or even single amino acid
but the use of fluorescent proteins sometimes lead to a conforma-
tional change of the target protein and large size of these fluorescent
proteins may alter the functionality of the target proteins (Zacharias
et al., 2002). Therefore, among site‐specific modifications, enzyme‐
based methods are robust, highly efficient and the major advantage
of enzyme‐based methods is that it requires mild conditions (Zhang
et al., 2018). Different classes of enzymes have been used, among
which Sortase A is a transpeptidase enzyme present in Gram‐positive
bacteria that helps in the recruitment of cell surface proteins
(Mazmanian et al., 1999; Ton‐That et al., 1999). It is a valuable bio-
chemical reagent as it is able to ligate the molecules together in vitro
(Jacobitz et al., 2017). Sortase A is a robust enzyme that is com-
mercially available and can be produced in moderate yields (>40 mg/
L) (Nuijens et al., 2019). The Sortagging reaction has been used for

Biotechnol Bioeng. 2021;118:4577–4589. wileyonlinelibrary.com/journal/bit © 2021 Wiley Periodicals LLC | 4577


4578 | KUMARI
different applications from protein cyclization to cell surface mod-
ification. The easy availability of the enzyme makes the approach
quite a popular tool in chemical biology.
In this review, we discuss the role of Sortase A in cell surface
modifications. We discuss the Sortase A‐based cell surface labeling in
both prokaryotic and eukaryotic cells. We also try to discuss the
advantages and shortcomings of the various methods.
2 | C E L L S U R F A C E MO D I F I C A T I O N S
Cell surface modifications are one of the significant tools to be used in
biomedical engineering. The cell surface modifications can be done by
various strategies, which are mainly divided into three categories de-
pending on the different approaches: (a) genetic‐based modifications, (b)
chemical‐based modifications, and (c) enzyme‐based modifications.
2.1 | Genetic‐based modifications
Genetic‐based modifications are well established and one of the
highly versatile methods for the cell surface modifications. The basic
step in genetic engineering is the insertion of exogenous genetic
material in the cell to express or suppress the expression of a specific
cell surface molecule (Kellam et al., 2003; Zhao et al., 2010). Many
studies were done that focused on the expression of receptors in-
volved in the cell surface recruitment and migration, to study the
ligand–receptor interaction, incorporated molecules to label the cells,
and many more (Howarth et al., 2005; Kato & Mrksich, 2004; Penn &
Mangi, 2008). Even though genetic engineering is quite a popular tool
but still there are few challenges related to regulatory and safety
issues (Custódio & Mano, 2016). The other issues associated with
genetic engineering approaches are the use of vectors for gene de-
livery. Viral vectors have higher transfection efficiency but they are
associated with immunogenic response and may lead to tumorigen-
esis. While other nonviral vectors have low transfection efficiency
(Liu et al., 2019), it leads to permanent modifications in cells, which
limits its applicability. Thus, other methods including chemical‐based
and enzyme‐based methods have been introduced.
2.2 | Chemical‐based modifications
Chemical‐based cell surface modification is the oldest and one of the
most used approaches. Various synthetic and natural molecules are at-
tached to the cell surface mainly by covalent bonding, hydrophobic in-
teractions, and electrostatic interactions (Figure 1). Covalent attachment
of chemical probes has dominance over noncovalent binding in terms of
handling, efficiency, and robustness (Sahoo, 2012).
Covalent conjugation is one of the straightforward approaches
that uses the exposed functional groups of proteins for grafting of
various chemical entities. The commonly used crosslinkers are the
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ET AL.
NHS (N‐hydroxyl‐succinimidyl ester), maleimide and pyridyldithiol
(Cabric et al., 2007; Digilio et al., 2010; Huang et al., 2015; Lee et al.,
2006, 2007; Torres & Gait, 2012). The NHS uses the exposed amine
group, maleimide‐conjugated molecules using exposed thiol group
while pyridyldithiol attaches through free thiol. Covalent modification
with a higher degree may cause significant alteration on the basis of
physiology, membrane mobility, and diffusion kinetics of the re-
engineered cells (Paulick et al., 2007; Rabuka et al., 2008; Teramura &
Iwata, 2010).
Electrostatic interactions use negatively charged cell surface and
positively charged polymers to modify the cell surface structure. The
main advantage of the electrostatic interactions is the protection of
the cells from sheer stress and immune response while biocompat-
ibility is a major concern to be used in therapeutics (Lee et al., 2018).
The other main strategy in the chemical‐based modifications is the
hydrophobic insertions and provides noninvasive modifications. Dif-
ferent lipophilic dyes (Dil, DiD, DiR, and DiO) that are currently
available in the market modifies cell surfaces through hydrophobic
interactions. Polyethylene glycols (PEGs) and polyvinyl alcohol (PVA)
are two amphiphilic polymers used in cell surface modifications. PEGs
interact with the lipid bilayer and also been used for surface plasmon
resonance (SPR) spectroscopy (Yamamoto et al., 2016). The ad-
vantage of hydrophobic insertion over other chemical‐based mod-
ifications is less toxicity and the ability of modified cells to resume
their normal activity (Carlos et al., 2009; Fujita et al., 2008; Miura
et al., 2006). The lipid‐conjugated biomolecules' fate is not fully un-
derstood and it requires further investigation (Inui et al., 2010).
In covalent conjugation, synthetic glycans are also used for the
modification of cell surfaces. Cell surface display of synthetic glyco-
lipids has been utilized to investigate the impact of specific glycan
structures on cell differentiation and behavior. Pulsipher et al. gener-
ated synthetic glycolipids and used liposomal delivery strategy, to
incorporate these glycolipids onto neuronal cell surfaces (Pulsipher
et al., 2014). Huang et al. conjugated heparin sulfate glycans to a
phospholipid tail (Huang et al., 2014), to control the differentiation of
embryonic stem cells. In another example, Paszek et al. generated
polymers modified with glycan structures found in natural mucin O‐
glycans (Paszek et al., 2014). The utility of synthetic glycolipids has
been limited by their rapid removal from the cell surface by inter-
nalization. The ability to generate long‐lived synthetic glycolipids will
expand the future applications of synthetic glycolipid engineering
(Woods et al., 2015).
2.3 | Enzyme‐based modifications
Enzymatic labeling techniques in living cells have gained attention
due to their high specificity, expeditious, mild conditions, efficient
reaction catalyzing “site‐specific” protein modification, and have
higher supremacy over nonenzymatic methods (Sahoo, 2012) (-
Figure 2). Different classes of peptidases, transferases, ligases, and
oxidoreductases are the major ones used in protein labeling. Among
all the classes, few are quite popular which are described in Table 1.
 10970290, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/bit.27935 by Universite Paris-Saclay, Wiley Online Library on [04/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KUMARI ET AL. | 4579

F I G U R E 1 Chemical labeling strategies for cell surface modification. Covalent conjugation uses N‐hydroxyl‐succinimidyl ester, maleimide
and pyridyldithiol for cell surface engineering. Electrostatic interactions using cationic and anionic polymers to allow modification by creating
different layers. Hydrophobic interactions uses polyethylene glycols and polyvinyl alcohol conjugates with alkyl tails embedded within cell
surface through hydrophobic interaction

In peptidase class, Sortase A and subtiligase are the most common
example. Another peptidase enzyme, subtiligase is the engineered en-
zyme derived from subtilisin present in the Bacillus amyloliquefaciens
(Wells & Estell, 1988). It catalyzes the reaction between acyl donor
peptide ester to the N‐terminal α‐amine of the acceptor of the peptide on
the N‐terminus. The major difference between subtiligase and subtilisin is
the change in its active site. S221 in the subtilisin is changed to cysteine in
the engineered enzyme that leads to shifts in the reaction from hydrolysis
to aminolysis (Abrahmsen et al., 1991). Even though it has ligase activity
but still excess of acyl acceptor fragment is required to suppress the
hydrolysis reaction, therefore, affecting the efficacy (Nuijenset al. 2019).
Microbial transglutaminase, farnesyltransferases, N‐
myristoytransferase, and phosphopantetheinyl transferase are the well‐
known enzymes in the transferases class. Microbial transglutaminase has
the ability to catalyze the reaction between γ‐carboxamide groups of the
glutamine residues and ε‐amino group of lysine and forms an amide bond.
It is extracted from Streptomyces mobaraensis. The enzyme activity is also
affected by the residues, which surround lysine. So, it is not able to
modify all glutamine or lysine residues (Spolaore et al., 2012). The ability
of the enzyme to modify the intrinsic residues is an advantage but be-
cause of its broad specificity, it is difficult to predict whether protein is
modified or not (Zhang et al., 2018). Farnesyltransferase, another

4580 | KUMARI
transferase recognizes CaaX motif and transfers an isoprenoid from its
substate farnesyl diphosphate. In CaaX, C stands for cysteine, a stands for
small aliphatic group and X decides the substrate specificity toward other
enzymes in the family (Jiang et al., 2018). As the enzyme uses only four
amino acids as its motif, it helps in minimizing the potential disruption of
target protein structure. The other enzyme from the transferase is N‐
myristoyltransferase, important for the lipid‐proteins cotranslationally/
post‐translationally modifications in eukaryotes (Beld et al., 2014). It
transfers the myristate from myristoyl‐CoA to the glycine at the N‐
terminal and aids in catalyzing an amide bond. Phosphopantethyeinyl
transferase catalyzes the reaction by transferring the Ppant (phospho-
pantetheine) group to PCP (peptiyl) or acyl carrier protein domain (Da
Silva Freitas et al., 2013). The enzyme is quite versatile in its applications
but the major disadvantage is its solubility (Zhang et al., 2018).
Ligases are the family that has the potential to catalyze the reaction
by fusing two large molecules. Tubulin tyrosine ligase belongs to this
family and helps in the tyrosine addition to the C‐terminal of tubulin by
catalyzing peptide bond using ATP (Ersfeld et al., 1993). Lipoic acid ligases
identify Lp1A acceptor peptide (LAP) and catalyze the reaction by at-
tachment of lipoate to lysine amino acid in LAP. While biotin ligase
conjugates the reaction by attachment of biotin onto protein using ATP.
All these enzymes catalyze protein modifications site‐specifically. Lipoic
acid ligase has also opened new possibilities as it can perform modifica-
tions at the intrinsic sites making the enzyme‐based modifications at-
tractive tool in protein engineering.
Oxidoreductases family is also one of the predominant family
uses in the protein labeling. The formylglycine generating enzyme
was first discovered by the Figura group while studying sulfatase
deficiency‐related diseases (Dierks et al., 2003). It performs the
modification of sulfatases type I by oxidation and hydrolysis of thiol
group of cysteine. Recombinant enzymes of the class were expressed
in Escherichia coli. Formylglycine generating enzymes‐mediated pro-
tein modifications have been developed as a commercial platform
called as SMARTag technology by Redwood Biosciences/Catalent.
Among all these enzymes, Sortase A, N‐myristoyltransferase, phos-
phopantetheinyl transferases, and formylglycine generating enzymes
are able to perform protein modification in E. coli (Zhang et al., 2018).
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ET AL.
F I G U R E 2 Systemic representation of
enzyme‐based labeling for cell surface
modifications. Enzymes recognize specific
substrate sequence and assist in the ligating
molecules onto the cell surface
Among all the enzymes, Sortase A, microbial transglutaminase,
and lipoic acids are the ones to perform labeling at any site, which are
the major advantages of these three enzymes when compared to
others. But as we already discussed, in the case of microbial trans-
glutaminase its activity is hindered by the surrounding residues to the
modification site. As it can modify glutamine or lysine residue, non-
specific modifications might be a problem. While lipoic acid ligase has
a recognition sequence of around 13 amino acids, which might dis-
rupt the structure of the protein, Sortase A has a small recognition
motif composed of only five amino acids.
3 | SORTASE A OF GRAM‐ P O S I T I V E
BAC TERIA
Sortase A (SrtA) is a member of the Sortases family, which aids in the
covalent recruitment of cell surface proteins on Gram‐positive bac-
teria. In 1990, the Schneewind group has discovered the activity of
Sortase enzymes. The same group also isolated the Sortase A enzyme
in 1999 (Ton‐That et al., 2000). After the discovery, the Sortase family
was vigorously studied. Sortase family has a total of six members
based on their recognition motif as well as their substrates and clas-
sified as A–F (Bradshaw et al., 2015; Clancy et al., 2010; Spirig et al.,
2011). Sortase A (SrtA) is the intensively studied and best known
among the Sortases family. In the early years of Sortase A discovery,
researchers realized that it could be an alternative target for anti-
bacterial therapy because it helps in the attachment of virulence
proteins on the cell surface of Gram‐positive bacteria (Mandlik et al.,
2008; Ton‐That et al., 2000). Inhibition of Sortase A will not create
pressure on bacteria as it is not essential for the growth of bacteria
(Guo et al., 2015). In 2004, Mao et al. demonstrated that SrtA has the
capability to perform in vitro protein ligation, which is more robust,
site‐specific, and reliable (Mao et al., 2004). The group performed the
peptide–peptide, protein–peptide, and protein–protein ligation. After
this study, the SrtA application in biomolecular engineering was ex-
tensively studied in the following years (Li et al., 2017; Reed et al.,
2020; Xu & Moyle, 2020).
 10970290, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/bit.27935 by Universite Paris-Saclay, Wiley Online Library on [04/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
KUMARI ET AL. | 4581

Staphylococcus aureus Sortase A (SaSrtA) has eight stranded β‐

barrel sheets with hydrophobic pockets for the substrate binding.

Grunewald et al. (2015); Pippig et al. (2014); Sunbul

Apart from SaSrtA, structure of other SrtA from Staphylococcus

Weeks and Wells (2018); Henager et al. (2016)

Ho and Tirrell (2016); Kulkarni et al. (2013)

pyogenes, Bacillus anthracis, Streptococcus agalactiae, and Strepto-

Dennler et al. (2014); Mero et al. (2016)

Pasqual et al., 2018; Wu et al. (2017)

Sueda et al. (2011); Kim et al. (2016)

Plaks et al. (2015); Gray et al. (2016)

Drake et al. (2014); Wu et al. (2009)

coccus mutans also has been reported (Khare et al., 2011; Race et al.,

Schumacher et al. (2017)

2009; Wallock‐Richards et al., 2015; Weiner et al., 2010). A common

Zhang et al. (2015)

characteristic of SrtA enzyme is the presence of short helix present

et al. (2008)
References

within β6/β7loop, which contacts the sorting signal LPXTG, upon

substrate binding (Chan et al., 2015; Suree et al., 2009).
SrtA recognize its substrate by LPXTG motif (X‐ any amino acid) and
performs the housekeeping role (Figure 3). Cell surface proteins are an-
chored by two‐step transpeptidation reaction (Bradshaw et al., 2015;
Clancy et al., 2010). First, the SrtA recognizes its motif and cysteine
residue in the active site nucleophilically attacks and cleaves between
threonine and glycine. Due to this, a thioacyl bond is established between
LPET‐SrtA. In the second step, the N‐terminal of oligoglycine present at
the lipid II of the cell‐wall nucleophilically attacks the thioacyl inter-
mediate, thus a peptide bond is formed between the carboxylic acid
Labeling terminal

N or C‐terminus

N or C‐terminus

group of threonine present in LPXT and the amino group of oligoglycine.

N‐terminus

N‐terminus
C‐terminus

C‐terminus

C‐terminus
Any site

Any site

Any site

By this mechanism, it recruits various cell surface proteins on the cell wall.
The main advantage of the SrtA is its ability to perform reactions in
Different enzymatic labeling methods with their recognition sequence, substrate and labeling terminal

nonnatural environment, its specificity toward its motif as well as it has

less impact on protein folding and functionality (Dai et al., 2019). But the
Tyrosine analogues Functionalized‐glycine

major pitfall of SrtA is its low catalytic efficiency, reversible nature of SrtA
reaction, and its Ca2+ dependence. Even though the low catalytic effi-
Lysine peptides, Primary amines

ciency and dependence on Ca2+ was partially resolved by the develop-

Myristicacid analogues

Lipoic acid analogues

Isoprenoid analogues
Glutamine Peptide

ment of SrtA variants having 140 times higher activity and variant, which
Biotin analogues
Peptide esters

Coderivatives

are Ca2+ independent (Antos et al., 2017). The reversibility of the reaction
Substrate

G(n)

can be resolved by using the excess of one substrate and shifting the
‐

equilibrium by removing the by‐products G‐XX (Antos et al., 2016;

Schmohl & Schwarzer, 2014). The latest strategy to tackle the reversibility
issue was resolved by the Antos group. They used metal‐coordinating
peptides. LPXTGGH is used instead of LPXTG as a recognition motif for
Sortase A. After cleaving by Sortase A, released GGH shows a higher
affinity towards Ni+2, therefore, deactivates the GGH peptide (Reed
Tub tag (VDSVEGEGEEEGEE)
Qtag (LLQG) Ktag (MKHKGS)

et al., 2020).
Recognition Sequence

GLNDIFEAQKIEWHE

wWhile performing the Sortase A‐based ligation, the accessi-

GFEIDKVWYDLDA
GDSLSWLLRLLN

bility of protein‐reactive motif to the enzyme is essential. Accessing

GNEASYPL

CXPXR
LPXTG

CaaX

the solvent availability NMR and protein crystal structure will be

‐

useful in assessing the success of sortagging. In the case of a steric

interface, a flexible linker can be used. A flexible linker can be kept
between the protein of interest and Sortase A reactive motif (Antos
et al., 2009).
Formylglycine generating enzyme
Phosphopantetheinyl transferase

4 | SORTASE A MEDIATED CELL SURFACE

Microbial transglutaminase

MODIFICA TION
N‐Myristoyltransferase

Tubulin tyrosine ligase

Farnesyltransferase

Lipoic acid ligase

Sortase, a special transpeptidase present in Gram‐positive bacteria

attaches many secreted proteins to the cell wall. The enzyme syn-
Biotin ligase
Subtiligase
TABLE 1

Sortase A

thesized recombinant protein with higher yield and stability. The

Enzyme

unique mechanism of Sortase A has opened a new avenue in protein

engineering, like protein–protein bioconjugation, circularization of
 10970290, 2021, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/bit.27935 by Universite Paris-Saclay, Wiley Online Library on [04/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4582 | KUMARI ET AL.

F I G U R E 3 Sortase A‐mediated recruitment of cell surface proteins on cell wall of Gram‐positive bacteria. Sortase A recognizes LPXTG motif
present at the C‐terminal of the cell surface protein. It forms thio‐acyl bond with the enzyme and the intermediate is resolved by the amine
which act as a nucleophile from the lipid II pentaglycine

FIGURE 4 Redecorated the cell surface of Staphylococcus aureus using small molecules having LPXTG motif via Sortase A (Nelson
et al., 2010)

proteins, peptide–nucleic acid hybrids preparation, labeling of the cell
surface, protein immobilization on a solid surface.
For several years, researchers are successfully using Sortase A for
decorating various living cell surfaces with LPXTG‐tagged small che-
mical probes, recombinant proteins and peptides, and polymers.
Nelson et al. documented that the cell surface of S. aureus can be
engineered using its endogenous Sortase A with a chemical or fluor-
escence probe (Figure 4). They have incubated LPXTG tagged biotin
with wild type S. aureus, resulting in covalent attachment of biotiny-
lated probe on the cell surface, which was confirmed by MALDI‐TOF
mass spectrum (Nelson et al., 2010). In another example, Manaskova
et al. modified the carboxy terminus of LPXTG with an amide group as
it may perturb the catalytic function of Sortase A. They have en-
gineered S. aureus cell wall with FITC tagged LPXTG amide and re-
ported that the incorporation of exogenous substrate of SrtA is
negatively correlated with SrtA mRNA expression and in a growth
phase‐dependent manner, which is peaked at the late stationary phase.
They also found that incorporation can be inhibited by hydroxylamine
(Maňásková et al., 2014). In a different study to further extend the
efficiency of Sortase‐mediated ligation, they have introduced three
types of modifications in SrtA substrate. The presence of positively
charged amino acid at C‐terminus increases the incorporation rate 20
folds, whereas methionine in place of aspartic acid increases in-
corporation threefolds. Same incorporation efficiency was observed
after addition of glycopeptide vancomycin with LPXTG motif, which
occurs at 500 folds low substrate concentration (Maňásková et al.,
2016). In search of an environmentally friendly feedstock for microbial
fermentation, Willson et al. have engineered Clostridium acetobutylicum
as a consolidated bioprocessing organism so that it can hydrolyze
lignocellulose and ferment the released sugar. By sortagging, they have
attached a cellulosome so that it could remain in close vicinity to the
released sugars (Willson et al., 2016).

KUMARI ET AL.
Apart from bacteria, researchers also tried to engineer cell sur-
face protein of mammalian cells using different suitable recombinant
proteins, fluorescent, or chemical probes. Popp et al. reported
mammalian cell surface labeling with the chemical probe by sortag-
ging. LPETG tagged CD40L was expressed on human embryonic
kidney (HEK) 293T cells. Cells were labeled with a biotinylated
pentaglycine probe by incubating in a sortase‐containing serum
medium. In this same study, they have also engineered influenza A/
WSN/33 NA with biotin (Popp et al., 2007). In a similar study, Tanaka
et al. has chosen osteoclast differentiation factor (ODF), a type II
membrane protein as their model cell surface protein. They have
transfected C terminus LPETG tagged ODF construct into HEK 293T
cell. The selective attachment of triglycine conjugated biotin and
EGFP on the cell surface occurs in the presence of 30 µM SrtA. The
labeling was confirmed by western blot using SAv‐HRP and anti‐
FLAG antibody (Tanaka et al., 2008). Nagamune and coworkers
showed that not only on C terminal, N terminal of cell surface protein
can also be labeled with SrtA. They have expressed a transmembrane
protein cyan fluorescence protein (CFP) on HEK 293T cell surface
and subsequently tagged with Alexa Fluor 647 by SrtA. They ana-
lyzed the labeling efficiency by using immunofluorescence staining
(Yamamoto & Nagamune, 2009).
To attach the labeling probe, cell surface protein has to be
modified with recognition motif, which may hamper the functional
activity of the protein. To overcome this problem, Park et al. de-
monstrated a one‐step method for labeling living cells without the
modification of membrane proteins. They have expressed opt‐
SrtAΔ59 containing a signal peptide from platelet‐derived growth
factor receptor (opt‐SrtAD59–TM) and labeled with EGFP and
TAMRA as fluorescent proteins and chemical dyes respectively (Park
et al., 2013).
Sortagging can also be a useful tool for modulating cell–cell in-
teraction. In their study, Tomita et al. directly incorporated a PEGy-
lated triglycine on murine EL4 thymoma cell line (E.G7 cells). Next,
they optimized the concentration of sortase (12 µM) and substrate
(50 µM) for maximum ligation efficiency followed by displaying Fc
domains of mouse IgG1 and IgG2a (Fc1‐LPETG and Fc2a‐LPETG) by
sortagging, which was confirmed by CLSM and FACS analysis after
immunostaining. Tagged cells were cocultured with the dendritic cells
at 37°C. Results showed a display of Fc2a‐LPETG increases the
phagocytosis rate by fourfold (Tomita et al., 2013). Swee et al. in-
corporated a single domain antibody on the surface of the activated
CD8 T cell by sortagging and redirected its cytotoxic activity speci-
fically toward Class II MHC positive B cell. The cell surface of the
parasite Toxoplasma gondi can also be labeled with a mouse Class II
MHC‐specific (VHH7) single‐domain antibody, which resulted in a
significant increase of B cell infection and subsequent lysis by this
parasite in comparison to non‐B cells (Swee et al., 2015). SML has
been exploited to overcome the risk related to the immune‐
suppressive treatment of the autoimmune disorder. To suppress
autoimmunity for a self‐antigen, Pishesha et al. made use of the
natural route of the destruction of RBC by phagocytic cells present in
the spleen. The LPETGG tag was introduced on the Kell protein of
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| 4583
RBC by CRISPR/Cas9 system. With SrtA around 9000 payloads (self‐
antigen) were attached to Kell‐LPETGG RBCs. This strategy shows
positive results in curing multiple sclerosis and type I diabetes in a
mouse model (Pishesha et al., 2017). With the help of SrtA‐mediated
transpeptidation, Tan et al. monitored intracellular trafficking of cell
surface receptors on a real‐time basis. By using the same labeling
method, the pH‐dependent dynamic distribution of OGR1 receptor
on the cell surface was visualized from the cell surface to various
intracellular compartments (Tan et al., 2018).
Research in different labs further extend the scope of sortase‐
mediated ligation, where they have engineered the virus capsid
protein. To observe the influenza virus budding in real time, Popp
et al. labeled two membrane proteins, hemagglutinin (HA) and
neuraminidase (NA), with biotin and Alexa Fluor 647 using extra-
cellular sortase. This study enables to monitor birth of new viruses in
infected cells (Popp et al., 2012). To make biological tools capable of
displaying a wide range of synthetic protein, Hess et al. engineered
M13 bacteriophage capsid protein pIII, pVIII, pIX with large molecules
like GFP and biotin with higher efficiency by using sortase A. They
have also shown that capsid proteins of the same bacteriophage can
display two or more different moieties (Hess et al., 2012). In a similar
approach, Schoonen et al. have experimentally proved that proteins,
fluorophore, and biotin can be attached selectively in cowpea
chlorotic mottle virus (CCMV) capsid protein by sortagging, which is
capable to encapsulate large cargos compared to VLPs from other
viruses (Schoonen et al., 2015). All the cell surface modifications have
been described in Table 2.
Wide varieties of substrates have been used by the Sortase A
in all these reports like capsid proteins of M13 bacteriophage,
capsid of CCMV, mammalian cell surface proteins, glycoproteins,
and many more. Using the HA protein (hemagglutination), the la-
beling efficiency was found to be 70%–80% while 68 ± 9% label-
ing efficiency was seen after 3 h at 37°C for pIII M13 capsid
protein (Hess et al., 2012; Popp et al., 2012). While using CCMV
capsid protein, the labeling efficiency was around 50%–60%
(Schoonen et al., 2015). In the case of mammalian cells, the re-
searchers evaluated the time for the labeling reaction. Park et al.,
group has found that 15 min is sufficient to monitor the labeling
(Park et al., 2013). Other groups used the ODF and suggested that
labeling could be monitored by 5 min incubation. They also
checked the labeling effect on the viability of cells and did not find
any effect. Therefore, this technique can be used in the future for
receptor‐trafficking and pulse‐chase labeling (Tanaka et al., 2008).
The labeling onto the cell surface has been done using en-
dogenous Sortase A as well as exogenous Sortase A. As most of the
gram‐positive bacteria have their own Sortase A (endogenous Sor-
tase A) on its cell wall, they do not require the enzyme/s from the
outside. As the presence of Sortase A has been limited to only gram‐
positive cells, the expressed and purified Sortase A (exogenous Sor-
tase A) can be used for modifying different surfaces and cells in which
the enzyme is absent. Although the expression and purification of the
enzyme hardly take 1–2 days, it will slightly affect the time for the
overall reaction.
 | 4584

TABLE 2 Cell surface modification of prokaryotic and eukaryotic cells using Sortase A
Cells Molecule Sortase used Work done Reference

Staphylococcus aureus Fluorescein, biotin and azide Endogenous First example of bacterial cell surface modification Nelson et al. (2010)

Staphylococcus aureus Chemically modified FITC LPETG‐ amide Endogenous Studied the effect of modified LPXTG domain and SrtA mRNA Manaskova et al. (2014;
expression on exogenous incorporation of chemical probe 2016)

Clostridium acetobutylicum Cellulosome Endogenous Engineered C. acetobutylicum to a consolidated bioprocessing Willson et al. (2016)
organism

Staphylococcus aureus and Peptides Endogenous Have seen the recruitment of peptide onto the cell surface of Kumari et al. (2020)
Enterococcus faecalis Gram‐positive bacteria leads to decrease in biofilm formation

HEK 293T cells Biotin, Alexa Fluor 647 and Osteoclast differentiate factor Exogenous Exploited sortase mediated ligation for labeling of cell surface Tanaka et al. (2008)
(ODF) labeled with Biotin, FLAG, HA tag, EGFP in protein in both carboxy and amino terminal.
different experiment

Hela cell EGFP Endogenous Demonstrated a method of cell surface labeling without attaching Park et al. (2013)
recognition motif for sortase A on surface protein.

Murine EL4 thymoma Fc region of mouse IgG1 and IgG2a Exogenous Used sortagging to study cell‐cell interaction. Tomita et al. (2013)
cell (E.G7)

Mouse hematopoietic cells, Biotin probe in all. Single domain antibodies at T cell surface Exogenous Redirected immunity towards a specific cell Swee et al. (2015)
yeast cells, 293 T
cells and Toxoplasma gondii

Human and mouse RBCs Peptides from disease‐relevant autoantigens like Exogenous Illustrated SML of self‐antigen on cell surface as an alternative of Pishesha et al. (2017)
MOG, OVA immune suppressive treatment
and insulin‐derived peptide

Leukocytes Hemagglutinin (HA) Exogenous Monitored intracellular trafficking of cell surface receptor on real Tan et al. (2018)
time basis.

Influenza A/WSN/33 strain Alexa Fluor 647, Biotin Exogenous Exemplify a sortase based ligation strategy to observe growth of Popp et al. (2012)
influenza virus.

M13 bacteriophage Biotin, GFP Exogenous Sortagging can be harnessed to engineer bacteriophage capsid Hess et al. (2012)
protein for higher labeling efficiency and to make them as a
CCMV viral capsid protein GFP, FITC Exogenous Schoonen et al. (2015)
tool to carry large molecules.
KUMARI
ET AL.

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KUMARI ET AL.
5 | C ONC LUS I ON
Various strategies have been developed and optimized for cell sur-
face modification. Chemical‐based approaches allow the ease in cell
surface modification without performing any genetic engineering on
the cells but it suffers from the off‐site modifications. But the de-
velopment of affinity‐based labeling performs the reaction in a con-
trolled manner (Tsukiji & Hamachi, 2014; Wang et al., 2011). As an
alternative to chemical‐based techniques, researchers are quite in-
terested in enzymatic‐based labeling because of the requirement of
mild conditions. Among these, Sortase A is quite a popular and well‐
studied enzyme for cell surface modifications. Sortase A has been
exploited since 2004 and researchers have found diverse applications
in different fields. The review gives useful insight on the Sortase A‐
mediated cell surface labeling. Sortase A is not only used for the
prokaryotic cells but has also been used for the modification of
mammalian cell surfaces, which makes it an interesting choice.
Sortase A‐based labeling can be used for both proteins and un-
natural moieties. Thus, we can play with a diverse number of mole-
cules. Sortase A cannot only perform the reaction in solution but it
can also catalyze the reaction even when immobilized on the solid
support (Steinhagen et al., 2013). They can perform modifications on
N‐terminal (Antos et al., 2009; Williamson et al., 2012), C‐terminal
(Antos et al., 2008; Popp et al., 2007; Popp & Ploegh, 2011), internal
loop regions (Guimaraes et al., 2011; Popp et al., 2009), which pro-
vides an additional advantage over other labeling methods. Time is
also an important parameter for this labeling. Despite being an or-
thogonal reaction, the cell surface labeling time of 45 min is enough
for robust labeling (Antos et al., 2009). Even though Sortase A has its
own limitations such as reaction reversibility and low catalytic ac-
tivity, different genetically engineered Sortase A are available to
tackle its low catalytic activity. Engineered sortases such as hepta-
mutant and pentamutant do not require Ca2+ ions. The other major
limitation, reversibility of the reaction is tackled by removal of some
of the reactants, using the excess of one of the reactants more as well
as use of metal peptide which has a high affinity toward GGG‐
fragment (Antos et al., 2017; Schmohl & Schwarzer, 2014). Different
substrates can be used with Sortase A such as peptides, membrane
proteins, soluble proteins, antibodies, bacterial toxins, and many
more (Popp et al., 2009; Popp & Ploegh, 2011; Sinisi et al., 2012;
Witte et al., 2013). Although the accessibility and the flexibility of the
region that is to be labeled is a crucial factor for successful trans-
peptidation. A flexible linker (Gly4Ser repeats) is needed but the exact
length of the linker cannot be determined and has to be determined
empirically. In most cases, efficient transpeptidation can be achieved
using the above solution but still to some extent the intrinsic struc-
tures of the proteins may inflict limitations. This case might be true
for labeling the internal part of the proteins, where modifying the
internal loop cannot be admissible.
The labeling can be combined with other methods like genetic‐
based to incorporate the LPXTG motif to the proteins. Click chem-
istry can also be used for the addition of the LPXTG motif or GGG at
the peptide and unnatural moieties.
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| 4585
We expect that endogenous and exogenous Sortase A will be
widely used to perform epitope‐grafting on bacterial cell surfaces as
well as cancer cells to reroute the immune system. The ease of this
method and combination of the sortase‐based labeling with other
labeling methods for a wide array of molecules and surfaces makes
the Sortase A more promising candidate in the field of protein en-
gineering. We envisage that the field of bioconjugation using Sortase
A will further evolve and will contribute immensely to biomaterial
synthesis.
ACKNOWLEDGME NT
We thank NIPER Guwahati and NIPER Kolkata for their constant support.
CONFLIC T OF INTERESTS
The authors declare there are no conflict of interests.
A UT H O R C O N T R I B U TI O N
The manuscript was written through the contribution of all the
authors.
DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were
created or analysed in this study.
ORC I D
Utpal Mohan http://orcid.org/0000-0002-0256-7305
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How to cite this article: Kumari, P., Bowmik, S., Paul, S. K.,
Biswas, B., Banerjee, S. K., Murty, U. S., Ravichandiran, V., &
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