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Recombinant cloning strategies for protein expression
Patrick HN Celie1, Annabel HA Parret2 and Anastassis Perrakis1
A variety of methods to create specific constructs for protein
expression, in a broad range of organisms, are available
nowadays. Restriction enzyme-free, ligation-independent and
recombinase-based cloning methods have enabled high-
throughput protein expression for structural and functional
studies. These methods are also instrumental for modification
of target genes including gene truncations, site-specific
mutagenesis and domain swapping. Here, we describe the
most common cloning techniques that are currently at hand for
recombinant protein expression studies, including a brief
overview of techniques associated with co-expression
experiments. We also provide an inventory of many of the
available reagents for the various cloning methods, and an
overview for some computational tools that can help with the
design of expression constructs.
Addresses
1
Division of Biochemistry, Netherlands Cancer Institute, 1066 CX
Amsterdam, The Netherlands
2
European Molecular Biology Laboratory, Hamburg Unit, Notkestrasse
85, 22607 Hamburg, Germany
Corresponding author: Celie, Patrick HN (p.celie@nki.nl)
Current Opinion in Structural Biology 2016, 38:145–154
This review comes from a themed issue on New constructs and
expression of proteins
Edited by Rob Meijers and Anastassis Perrakis
For a complete overview see the Issue and the Editorial
Available online 5th July 2016
http://dx.doi.org/10.1016/j.sbi.2016.06.010
0959-440X/# 2016 Elsevier Ltd. All rights reserved.
Introduction
Molecular cloning techniques have advanced dramatical-
ly since the discovery of the first restriction endonu-
cleases. Recombinant DNA technology is nowadays
considered a routine practice. DNA isolation and ampli-
fication, Polymerase Chain Reaction (PCR), molecular
cloning, and — more recently — genome editing have
become standard procedures.
In the early 1970s, discovery of the restriction enzymes
HindIII [1] and EcoRI [2] led to a significant break-
through in the development of recombinant DNA tech-
nology and pioneered the first cloning experiments
transferring DNA fragments from one bacterial strain
to another, using a carrier plasmid [3]. Cloning of a
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complete bacterial gene followed [4], and the discovery
that DNA from different species can be cloned and
propagated in another species [5] signified the beginning
of the heterologous recombinant gene expression tech-
nologies. In the 1980s, the breakthrough invention of
PCR to amplify DNA fragments in vitro [6], allowed
advanced cloning methodologies to emerge. Routine
synthesis of large DNA fragments of specific sequence
[7] that became mainstream and affordable in the last
decade, was another landmark in laboratory practice. In
this review, the most popular cloning techniques that are
of direct relevance to protein expression will be presented
and discussed.
Restriction and ligation-based cloning
At present, the restriction enzyme repertoire consists of
more than 200 highly specific and commercially available
proteins. Likewise, many diverse cloning vectors exist
that enable ligation of DNA fragments digested with
specific restriction enzymes into their multiple cloning
site (MCS), consisting of unique restriction sites
(Figure 1). However, cloning can become complicated
when genes contain internal restriction sites that are also
present in the MCS. In addition, when multiple expres-
sion constructs have to be made, for example, for protein
production in various host organisms or when different
affinity and solubilizing tags need to be tested, compati-
bility between the MCS of different vectors is an issue. In
addition, many restriction enzymes often do not cleave
linear PCR products well, but prefer circular DNA. Fi-
nally, ‘seamless’ cloning is not always possible. The
selected restriction site(s) may introduce additional
nucleotides into the coding sequence, causing a scar in
the gene which leads to incorporation of non-native
amino acid residues into the expressed protein. Various
solutions have been invented to tackle some of these
cloning issues. Multiple-host vectors have been designed
to allow protein expression from the same construct in
E. coli, insect, and mammalian cells (pTriEXTM vectors;
EMD Millipore). To enhance cleavage specificity of
DNA fragments, a deoxyinosine nucleotide can be incor-
porated in flanking DNA sequences, to allow cleavage by
Endonuclease V [8]. Type II restriction endonucleases,
which cleave outside their recognition sequence marked-
ly facilitate seamless cloning [9]. This approach has been
adopted by Golden Gate cloning [10–12] and is commer-
cialized by NEB. TOPO1-TA (sub) cloning (Thermo
Fisher Scientific) is a restriction-independent cloning
method. It takes advantage of the specific feature of
the Taq polymerase, which adds a 30 overhanging adenine
(A) base to a double-stranded DNA fragment. This
allows efficient topoisomerase-assisted ligation of a
Current Opinion in Structural Biology 2016, 38:145–154
146 New constructs and expression of proteins

Figure 1

Restriction-based cloning
MCS
E. coli
Vector Restriction
Endonucleases Expression
Clone Transformation
Ligase

restriction restriction Restriction +

site 1 site 2 Endonucleases
PCR product or
vector containing gene

Ligation-independent cloning
E. coli E. coli
LIC sequence

Vector Linearisation
Annealing
T4 treatment
Transformation

Lic sequence T4 treatment +

Lic sequence
PCR product

Recombination-based cloning (Gateway®)

attR1
attP1
ccdB - lethal gene
Donor
ccdB - lethal gene Destination E. coli
Vector attR2
Vector attP2 attB1

Transformation
+ BP
clo + attL1
LR clonase Expression
Clone
attB2

attB1
nas
e
attB2
PCR product or Entry attL2
vector containing gene Clone

Current Opinion in Structural Biology

Schematic representation of three different cloning techniques. Restriction-based cloning requires restriction endonucleases to create compatible
overhangs in vector and insert which can be joined by a ligase. In ligation-independent cloning (LIC) [14], compatible overhangs in insert and
vector are created by exonuclease activity of T4 polymerase and the ligation of the fragments occurs inside E. coli upon transfection of the
fragments. Recombination of two DNA fragments, like in the Gateway1 system [26,27,42], requires specific sequences (att) within insert and
vector, which are recognized by the recombinase enzyme mixtures (Clonase). Within the Gateway1 system, typically a subset of entry clones is
created which can be used for subsequent cloning of the insert into destination (expression) vectors.

PCR fragment into a linearized vector comprising a
thymidine (T) overhang at the 50 end. Directionality
issues, as well as the high cost of vectors covalently linked
to topoisomerase, have compromised the popularity of
this solution; it has often been used, however, to transfer a
PCR product to an ‘intermediate’ vector, avoiding issues
with restriction enzymes being inefficient in cutting PCR
products.
Notably, the USER cloningTM strategy from NEB [13] is
unidirectional, although it requires a specific enzyme
mixture, and DNA fragments have to be amplified with

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primers containing a deoxyuracil (dU) base preceded by a
specific 7 bp sequence at the 50 end. This sequence is
recognized and cleaved by the USER enzyme mix, leav-
ing a fragment with an 8 bp 50 overhang which can
subsequently be cloned into a specific (linearized) vector
containing complementary overhangs.
Ligation-independent cloning
When DNA synthesis via PCR became routine in the
early 1990s, cloning technologies that did not require
restriction enzymes or ligase started to emerge [14–16].
These methods aimed to improve cloning efficiency,
reduce cost and minimize cloning time. The demand
for large scale open reading frame (ORF) clone libraries
became clear with the rise of Structural Genomics and
Proteomics consortia. These centers aimed to elucidate
protein structures and interactions, building on sequences
from Genome projects. At that time, the advent of syn-
thetic gene technologies and Ligation-Independent
Cloning (LIC) [6,14] came strongly into play. In LIC
cloning, both PCR fragment and linearized vector are
flanked by complementary sequences of 12–18 bp, de-
void of one of the four nucleotides (dATP, dCTP, dGTP
or dTTP) (Figure 1). Single stranded DNA overhangs are
created by separate incubation of the PCR fragment and
vector with T4 polymerase, in the presence of only the
particular nucleotide that is absent in the overhang se-
quence. The exonuclease activity of the polymerase,
which normally serves a proofreading function, removes
nucleotides from the 30 site until it encounters the specific
nucleotide that has been added to the reaction, effective-
ly creating a long single-stranded overhang. The PCR
fragment and vector are mixed and the complementary
cohesive ends anneal and, owing to the large overhangs,
are ligated inside the E. coli cell (Figure 1). As LIC
cloning is easy to implement and requires relatively cheap
reagents, it became a popular technique. Many LIC
vectors containing specific complementary sequences
have been designed for (high-throughput) cloning, pro-
tein expression [17–20] and library construction [21] and
are also commercially available (Merck Millipore). The
limitation for specific complementary sequences has been
overcome in case of Sequence and Ligation-Independent
Cloning (SLIC) [22]. The SLIC protocol is highly similar
to that of LIC, with the exception that somewhat larger
complementary sequences (20–40 bp) are necessary for
optimal cloning efficiency. However, the advantage is
that there is no restraint for absence of a specific nucleo-
tide, because T4 polymerase treatment is done in the
absence of nucleotides, resulting in more extensive single
strand overhangs. The annealing step can be enhanced by
addition of RecA protein, in particular when low amounts
of DNA are used.
The concept of (S)LIC cloning, that is exonuclease
treatment followed by annealing and spontaneous liga-
tion of single stranded DNA overhangs within the cell,
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Recombinant cloning strategies Celie, Parret and Perrakis 147
forms the basis of a number of related methods. Gibson
et al. combined the benefits of LIC and SLIC into a
cloning procedure that requires short complementary
DNA stretches (15 bp) without any sequence restrictions
[23]. However, polymerase and ligase have to be added
after treatment with T5 exonuclease, in order to fill up the
DNA gaps caused by extensive exonuclease processing
by the enzyme. The reagents for Gibson cloning are
commercially available as a cloning kit from NEB (Gibson
Assembly1 cloning kit). Other companies offer similar
cloning kits, including In-Fusion1 Cloning (Takara Bio-
tech) [24], GeneArt1 Seamless Cloning, (ThermoFisher
Scientific), Cold Fusion1 Cloning (SBI), Fast Seamless1
Cloning (Dogene) and CloneEZ1 (Table 1)
Recombination-based cloning
Several site-specific techniques using recombination
have been developed to facilitate high-throughput clon-
ing. These methods all require the presence of entry (or
donor) plasmids and recombination into a final destina-
tion vector (Figure 1). The entry clones are typically
obtained following directional blunt-end cloning, as in
the univector plasmid–fusion system (UPS) [25] and the
Gateway1 system [26,27] or by restriction-based cloning
(Gateway1 and Mating-Assisted Genetically Integrated
Cloning (MAGIC) [28]). Entry clones can also be
obtained from a (commercial) library (e.g., Gateway1)
and these clones can serve as the basis for subsequent
cloning into dedicated vectors, depending on the biologi-
cal purposes (protein over-expression, in vivo detection,
pull-down assays, etc.). The Gateway1 cloning system is
based on in vitro recombination, using the integration of
lambda bacteriophage into the E. coli genome by the
Integrase enzyme and att recombination sites [26]
(Figure 1). For this system, PCR fragments containing
attB sites can also be recombined into donor vectors [29]
or directly into destination vectors [27].
A method that enjoys increasing popularity is SLiCE
(Seamless Ligation Cloning Extract) cloning [30,31].
It is based on ex vivo recombination and uses E. coli cell
extract containing the native enzymes, instead of a re-
combinant enzyme mixture for cloning. This simple
method requires at least 15 bp flanking the PCR frag-
ment, complementary to the insertion sequence within
the vector. The recombination mechanism is not exactly
clear since it appears to be RecA-independent, but the
efficiency is increased by using an extract prepared from
an E. coli strain expressing proteins involved in the Red
recombination system [30].
PCR-based cloning
PCR-based cloning methods typically require whole-
plasmid amplification of vector and insert together, but
do not require restriction endonucleases, recombinase or
ligase. Polymerase Incomplete Primer Extension (PIPE)
cloning [32] is based on the fact that during PCR reactions
Current Opinion in Structural Biology 2016, 38:145–154
148 New constructs and expression of proteins
incomplete extension of fragments occurs, resulting in a
mixture of PCR products comprising different lengths of
single stranded overhangs. DNA insert and vector, which
share 14–17 bp complementary overhangs, are both am-
plified during (separate) PCR reactions and the products
are mixed and transformed; this method has been suc-
cessfully applied to clone 448 targets for structural geno-
mic purposes [32]. Another method is the Restriction-
Free (RF) cloning procedure [33,34] or Overlap Exten-
sion PCR cloning [35], based on the QuickchangeTM
method (Stratagene). A PCR fragment of interest, flanked
by a 24 bp sequence overlapping with the insertion site of
the destination vector, is synthesized; this serves as a
mega-primer that is incubated with the circular destina-
tion vector to obtain the complete construct in a second
PCR reaction. After completion of the PCR reaction, the
(methylated) template vector is digested by the restric-
tion enzyme DpnI to reduce background colonies upon
transformation of the DNA. This method depends on
efficient DNA polymerases like pfuTurbo1 (Agilent),
Q51 (NEB) or Phusion, which have both high processivity
and proofreading capacity. This is essential for obtaining
good quality and error-free DNA. An improvement to that
protocol is the Transfer-PCR (TPCR) system, in which
the separate fragment amplification and insertion reac-
tions are merged into a single PCR reaction which con-
tains donor vector, destination vector and primers [36]. In
the Circular Polymerase Extension Cloning (CPEC)
method [37], linearized vector (e.g., obtained by PCR)
can be used to integrate a PCR fragment, which elim-
inates the requirement for DpnI treatment.
In PCR-based cloning, the final construct is typically
obtained by linear product amplification, which reduces
the chance of introducing DNA errors during the reaction
when compared to traditional PCR reactions where DNA
is amplified exponentially. However, linear amplification
results in relatively low product yield. If this is an issue,
exponential mega-priming (EMP) cloning can be an
alternative. This is a RF-based system in which the
PCR product is flanked at only one terminus by a 20–
25 bp complementary sequence and the (linear) product
is exponentially amplified [38]. A final DNA ligation step
is required to create a circular plasmid. Another method to
assemble DNA via PCR is the ligase cycling reaction
(LCR) where single-stranded ‘bridging’ DNA oligonu-
cleotides are used to connect fragments in a multi-step
PCR reaction [39].
Generation of mutation and deletion
constructs
To understand protein function and structure it is often
necessary to explore protein variants like point mutations,
deletions, domain swapping and truncations. For cloning
of fragments or (consecutive) domains, any of the cloning
methods can be applied once the DNA fragment of
interest is amplified by PCR. However, when internal
Current Opinion in Structural Biology 2016, 38:145–154
mutations or deletions have to be introduced, different
strategies may have to be applied. The QuickChangeTM
strategy (Agilent) is probably the most common site-
directed mutagenesis method to delete or substitute
several bases within a short stretch of DNA (<50 bp)
using a single PCR reaction. It requires two complemen-
tary primers that contain the mutation(s) in the middle of
the sequence and circular target plasmid DNA. The
mutation(s) are flanked by a stretch of 10–15 bases at
both sides, which anneal to the template DNA. Following
cycles of DNA annealing and extension, the entire plas-
mid DNA containing the desired mutation is amplified
(Figure 2). Template DNA is selectively digested by
DpnI endonuclease to reduce background transforma-
tion. This procedure can also be executed without a
kit, using separate reagents, including polymerase, pri-
mers and DpnI enzyme. A variation of this protocol is
provided by the Q51 Site-Directed Mutagenesis kit
(NEB), which uses ‘back-to-back’ primers rather than
complementary primers. To introduce mutations simul-
taneously at multiple sites, the QuikChange Multi Site-
Directed Mutagenesis Kit can be used [40]. It is based on
the same principle as the QuickChangeTM method, ex-
cept that an enzyme blend additional to the polymerase is
required to seal the nicks between the created fragments.
This method is also suited for the creation of randomized
mutation libraries using degenerate primers [40]. Similar
to the QuickChangeTM method, the PIPE cloning strat-
egy can be used to create substitutions and internal
deletions within a single PCR reaction. The mutation/
substitution has to be present in the overlapping region
(15 bp) of both forward and reverse primer [41].
If larger DNA fragments need to be inserted or replaced,
the restriction-free PCR-based cloning procedures de-
scribed above can be applied (Figure 2). Deletions up
to 2.4 kb and insertions up to 3.5 kb have been success-
fully introduced [34]. RF cloning has also been advanced
into a multi-site mutagenesis method: multiple DNA
fragments, each comprising a flanking sequence of more
than 20 bases which overlap with the adjacent fragment,
are combined together with the target DNA into one PCR
reaction. The final product contains the concatenated
fragments inserted into the plasmid. Similarly, the
LCR method described above, has been used to assemble
up to 20 DNA fragments in a single-step reaction [39]
(Figure 2). A particular note concerning all of PCR-based
methods, is the usage of high-fidelity enzymes with
reliable proofreading capacity to avoid unwanted muta-
tions during the amplification of the DNA that could
affect protein expression.
In addition to PCR-based methods, exonuclease-based
cloning methods can be used to modify DNA constructs
at multiple sites [42]. Multiple fragments, which together
form the complete new construct, are created by PCR,
using primers that contain the respective mutations and
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 Recombinant cloning strategies Celie, Parret and Perrakis 149

Figure 2

“Quickchange” mutagenesis Restriction-free / Overlap Extension PCR

New
Mutated Mutated clone
Mutagenic plasmid plasmid
Primers PCR PCR
Vector
PCR DpnI DpnI
Template

Template Template
+ vector
PCR mega-primer

Ligase Cycling Reaction

“Leftover”
“-” strand
Cut
Vector
denature “Patched”
“+” strand
ligase Ligated + + denature “Patched”
new clone
ligase Ligated
new
anneal “+” strand anneal
Insert 1 Insert 2 clone

+
Insert 1 Insert 2
single stranded primers

Current Opinion in Structural Biology

Three PCR-based methods to introduce mutations, deletions and insertions into expression constructs. QuickchangeTM (Agilent) can be used to
introduce short substitutions, insertions or deletions by using complementary primers containing the respective modification within a single PCR
reaction. RF cloning [33,34] or Overlap Extension PCR cloning [35] can be used to introduce a (large) fragment into an existing plasmid. The
fragment is flanked by sequences that are complementary to the insertion sites within the vector. Upon denaturation, vector and insert can anneal,
followed by synthesis of the complementary DNA strand during a PCR reaction. LCR [39] can be used to join (multiple) fragments during a series
of denaturation, annealing and ligation cycles. Single-stranded DNA oligos are used to connect two separate fragments, which will be
subsequently joined by a thermostable ligase.

are flanked by at least 10 bp which are homologous to the Protein co-expression

adjacent fragment in the new construct. The fragments
are joined upon exonuclease treatment, using any of the
commercial kits like Infusion1, GeneArt1 Seamless
Cloning and Assembly or Gibson AssemblyTM Master
Mix before the DNA is transformed into E. coli.
A challenge in the field of structural biology is the produc-
tion of protein complexes at amounts amenable for
functional and structural studies. Although in vitro recon-
stitution of such complexes can succeed, it is often a time-
consuming and complicated process that generally

Figure 3

Multiple vectors Single vector Single vector

Multiple promoters One promoter
Promoter
RBS
Gene of interest

Resistance marker

Origin of replication
Current Opinion in Structural Biology

Co-expression strategies for production of multiple proteins/protein complexes. Three different methods exist for co-expression of proteins: First,
multiple vectors can be used to introduce genes into host cells. Typically, each vector contains a specific gene of interest and different antibiotic
selection marker and, if more than two plasmids are introduced, also dissimilar origins of replication. Second, poly-cistronic constructs enable
expression of multiple genes from a single vector. All genes are under control of one single promoter and each gene is preceded by a ribosomal
binding site (RBS). Third, co-expression of proteins from a single vector. In contrast to poly-cistronic expression, each gene is under control of a
separate promoter. Promoters can be identical for each expression cassette or, alternatively, different promoters can be used for each individual
gene.

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150 New constructs and expression of proteins

requires additional purification steps [43]. Moreover, this
approach is unlikely to succeed when individual proteins
are unfolded or insoluble in absence of specific protein
partners, as is often observed upon expression of individual
subunits of cellular complexes. To date, the method of
choice to produce multiprotein complexes is based on in
vivo assembly of the individual protein subunits following
heterologous expression of multi-gene constructs [43–46].
Several comparative studies suggest that there is no pre-
ferred strategy to achieve multi-protein expression [45,47].
Co-expression can be achieved by three methods
(Figure 3): First, using multiple vectors, each carrying
one gene of interest, where complex formation is driven
by co-transforming the expression vectors; second, using
a single vector comprising multiple expression cassettes,
each under control of a separate promoter; third, using one
vector that creates a poly-cistronic mRNA under the con-
trol of a single regulatory element, with several genes, each
preceded by aribosome binding sites (RBS). The latter
approach requires careful design of the cloning strategy,
taking into account that the order and number of the genes
within the poly-cistronic transcript as well as the location of
the tag can dramatically influence the yield of the recon-
stituted protein complex [44,47,48]. In any case, the se-
lected cloning strategy to accomplish co-expression should
be flexible and parallelizable to allow a combinatorial
approach that is often crucial for the success of multi-
protein expression. Until recently, classical restriction en-
zyme-based cloning was the method of choice for assembly

Table 1

Cloning methods for creating protein expression constructs

Cloning strategy Enzymes required Cloning site/sequence Refs, Website

Vector Fragment
Restriction enzyme/ligation-based cloning
a,b,c,d,e
Restriction Restriction enzymes, T4 DNA Ligase Restriction site Restriction site
a
Golden Gate cloning Type II endonuclease, T4 DNA Ligase Restriction site Restriction site
a
USER USER enzyme mix 8-12 bp dU in pimer
Ligation-based cloning
b
Topo1-TA T4 DNA ligase 30 T-overhang 50 A-overhang
Exonuclease -dependent cloning/single strand DNA annealing
LIC T4 DNA polymerase >12 bp LIC sites >12 bp LIC sites [14]
SLIC T4 DNA polymerase, RecA No specific 20–40 bp [22]
c
In-Fusion1 In–Fusion HD cloning enzyme mix No specific 15 bp [24]
11 a
Gibson assembly T5 Exonuclease, Phusion DNA polymerase, No specific 15–80 bp [23]
Taq ligase (Or proprietary enzyme mix)
b
Geneart1 seamless cloning Proprietary enzyme mix No specific 15 bp
Recombination-dependent cloning
b
Gateway12 Proprietary Clonase enzyme mixtures 25 bp att site 25 bp attB site [26,27,42]
UPS Cre Recombinase 34 bp loxP site 34 bp loxP site [25]
MAGIC 3 I-Sce endonuclease, Reda Redb and Gam 50 bp sequence, 50 bp sequence, [28]
(in vivo) I-SceI site I-SceI site
SLiCE 4 E. coli extract No specific 15–52 bp [30]
PCR-based cloning/single-strand DNA annealing
PIPE DNA polymerase No specific 14–17 bp [32,41]
RF DNA polymerase, DpnI No specific 24–30 bp [33,34]
TPCR DNA polymerase, DpnI No specific 30 bp [36]
CPEC DNA polymerase No specific 15–25 bp [37]
EMP 5 DNA polymerase, T4 PNK, DpnI No specific 20–25 bp [38]
T4 DNA Ligase
LCR 6 Thermostable ligase No specific [39]

Remarks:
1
Ligation required.
2
Many destination vectors available.
3
Specific donor and recipient E. coli strains required.
4
Mechanism not completely clear.
5
Final ligation step required.
6
Melting temperature of bridging oligo > 60 8C.
List of vendor web sites:
a
https://www.neb.com/.
b
https://www.thermofisher.com/.
c
http://www.clontech.com/.
d
http://www.promega.com.
e
https://lifescience.roche.com/.

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 Recombinant cloning strategies Celie, Parret and Perrakis 151

of poly-cistronic vectors for heterologous expression of
recombinant protein complexes [44,49–51]. Generally, this
strategy implies that each coding sequence is cloned in a
‘donor’ plasmid flanked by unique restriction sites. Fol-
lowing a stepwise approach, multiple coding sequences can
then be concatenated in ‘acceptor’ vectors. Although a
number of convenient vector systems have been developed
for E. coli and insect cells, the major drawback of this
technique is the need for specialized vector systems and
the obvious limitation of using restriction enzymes
(Table 1).
Many of the recombinase and PCR-based methods de-
scribed above can be easily adapted or combined to
generate multi-gene constructs and are now widely used
in the field of synthetic biology [52,53]. However, these
techniques have some drawbacks for structural biologists
given that they were not specifically designed for the
generation of expression constructs.
based on yeast homologous recombination [57]. A varia-
tion of this method, the any-gene-any-plasmid (AGAP)
cloning, has recently been described and although not
widely applied, the authors highlight the general appli-
cability of this method as a cost-effective efficient method
for multiprotein reconstitution in microbial and verte-
brate expression systems [58].
The assembly of large and highly repetitive protein
coding genes can be particularly challenging. In order
to address this, a plethora of recombination-based tech-
niques have been developed in the field of synthetic
biology, such as Golden Braid for protein expression in
plants [59,60], USERec [61], unique nucleotide se-
quence (UNS)-guided assembly [62,63] and advanced
Gibson assembly methods [64]. LIC-based cloning meth-
ods are equally popular for multi-gene assembly of highly
repetitive genes for example in case of transcription
activator-like effector proteins [65].

Although originally tailored towards expression of eukary-
otic multiprotein complexes in insect cells [54], the
ACEMBL technology is now well established in the
structural biology community given its wide applicability
and automatable workflow [55,56]. An interesting alter-
native approach amenable to multi-gene assembly is
Choosing a suitable cloning strategy
The choice for a particular cloning strategy depends on
many factors, including the number of genes to be
expressed, the variation in (truncation) constructs to be
made, the range of vectors and tags to be used and the
different expression systems to be tested. In addition, also

Table 2

Useful resources for cloning projects

Tool Website Reference

Cloning tools
Protocols for molecular cloning
Protocols for molecular cloning
Collection of cloning resources
Construct design tool
DNA sequence analysis
Prediction of Translation initiation
rates and optimization of mRNA
sequence
Analyze sequence for restriction sites
Complete list of restriction enzymes
Plasmid annotation
Useful DNA calculator
Codon usage tables
Rare codon calculator
Rare codon calculator
Online codon optimization tool
Online codon optimization tool
Web portals
Portal for bioinformatic tools
Portal for bioinformatic tools
Human gene database
Complete software packages
(freeware)
GenomeCompiler
SnapGene Viewer
SerialCloner
ApE
http://www.protocol-online.org/prot/Molecular_Biology/Molecular_Cloning/index.html
http://www.molbiotools.com/
https://www.neb.com/tools-and-resources
https://xtal.nki.nl/ccd/ [70]
https://salislab.net/software/ [71,72]
http://www.restrictionmapper.org/
http://rebase.neb.com/cgi-bin/asymmlist
http://wishart.biology.ualberta.ca/PlasMapper/ [73]
http://nebiocalculator.neb.com
http://www.kazusa.or.jp/codon/
http://nihserver.mbi.ucla.edu/RACC/
http://www.codons.org/index.html
http://www.jcat.de/ [74]
http://genomes.urv.es/OPTIMIZER/ [75]
http://mobyle.pasteur.fr/cgi-bin/portal.py
http://www.expasy.org/
http://www.genecards.org/
http://www.genomecompiler.com
http://www.snapgene.com
http://serialbasics.free.fr/Serial_Cloner.html
http://bioweb.biology.utah.edu/jorgensen/wayned/ape/

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152 New constructs and expression of proteins
laboratory habits and financial and instrumentation
resources will influence the choice of a particular cloning
strategy. There is probably not a single method that is
superior to all cloning and expression studies. A compari-
son of three LIC strategies, including Overlap Extension
Cloning (OEC), PIPE cloning and SLIC, illustrated that
all three methods show high cloning efficiencies for
inserts < 1.5 kb, while OEC is less efficient for frag-
ments > 1.5 kb compared to the other two systems
[66]. In addition, particular care should be taken with
PCR-based cloning methods which rely on amplification
of entire plasmids, like OEC. During PCR amplification,
errors can be introduced within the plasmid backbone
that could affect protein expression levels even though
the gene sequence is correct. The creation of a cloning
strategy blueprint, including primer design, vector selec-
tion and protocol setup can be quite laborious, however,
there are numerous websites that are very useful to
explore before preparing your constructs (Table 2).
Expression of sufficient amounts of soluble protein for
functional and structural studies often depend on factors
which are beyond the chosen cloning strategy: choice of
expression system, expression conditions and vector to-
pology. However, the design of the vector should be taken
into account when selecting a cloning strategy, namely,
which promoter to use, which tag and at what position (N
or C-terminal), which origin of replication and so forth.
Although many vectors are already optimized for efficient
protein production, differences in expression levels are
still observed between vectors, even if tag and insert are
identical [45]. To promote swapping of different vector
elements, a modular design of vector modules has been
first described in 1996 [67] and has later been developed
and formulated into BioBricks by Tom Knight [68]. These
BioBricks can be different modules like promoter
sequences, ribosomal binding sequences (RBS), antibiotic
resistance markers and origin of replications. These
sequences are flanked by specific endonuclease recogni-
tion sites in order to promote efficient building of new
vectors [69]. However, also PCR-based methods and
Gibson cloning are used to create vectors from BioBricks.
Together, the development of new expression tools, the
wide selection of cloning methodologies and the avail-
ability of codon-optimized synthetic genes, expand the
versatility in protein expression methods and facilitate
screening of optimal protein production conditions.
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