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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 highthroughput 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, 38:145154

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 dramatically since the discovery of the first restriction endonucleases. Recombinant DNA technology is nowadays
considered a routine practice. DNA isolation and amplification, 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 breakthrough in the development of recombinant DNA technology 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 technologies. 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 expression 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, compatibility 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. Finally, 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 incorporated in flanking DNA sequences, to allow cleavage by
Endonuclease V [8]. Type II restriction endonucleases,
which cleave outside their recognition sequence markedly facilitate seamless cloning [9]. This approach has been
adopted by Golden Gate cloning [1012] and is commercialized 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, 38:145154

146 New constructs and expression of proteins

Figure 1

Restriction-based cloning
MCS

E. coli

Restriction
Endonucleases

Vector

Ligase

Transformation

Restriction
Endonucleases

restriction
site 2

restriction
site 1

Expression
Clone

PCR product or
vector containing gene

Ligation-independent cloning

E. coli

LIC sequence

Linearisation
T4 treatment

Vector

Annealing
Transformation

T4 treatment

Lic sequence

E. coli

Lic sequence

PCR product

Recombination-based cloning (Gateway)

attR1

attP1
ccdB - lethal gene

Donor
Vector

attP2

BP

Destination
Vector

clo
n

as

attB1

ccdB - lethal gene

E. coli

attR2
attB1

LR clonase
attL1

Expression
Clone

Transformation
attB2

attB2

PCR product or
vector containing gene

Entry
Clone

attL2

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
Current Opinion in Structural Biology 2016, 38:145154

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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Recombinant cloning strategies Celie, Parret and Perrakis 147

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, leaving 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 [1416].
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 synthetic 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 1218 bp, devoid 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 sequence. 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, effectively 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, protein expression [1720] 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 (2040 bp) are necessary for
optimal cloning efficiency. However, the advantage is
that there is no restraint for absence of a specific nucleotide, 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 ligation of single stranded DNA overhangs within the cell,
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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 Biotech) [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 cloning. These methods all require the presence of entry (or
donor) plasmids and recombination into a final destination vector (Figure 1). The entry clones are typically
obtained following directional blunt-end cloning, as in
the univector plasmidfusion 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 biological 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 recombinant enzyme mixture for cloning. This simple
method requires at least 15 bp flanking the PCR fragment, 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 wholeplasmid 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
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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 1417 bp complementary overhangs, are both amplified during (separate) PCR reactions and the products
are mixed and transformed; this method has been successfully applied to clone 448 targets for structural genomic purposes [32]. Another method is the RestrictionFree (RF) cloning procedure [33,34] or Overlap Extension 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 destination 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 restriction 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 reactions are merged into a single PCR reaction which contains 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 eliminates 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 oligonucleotides are used to connect fragments in a multi-step
PCR reaction [39].

mutations or deletions have to be introduced, different

strategies may have to be applied. The QuickChangeTM
strategy (Agilent) is probably the most common sitedirected mutagenesis method to delete or substitute
several bases within a short stretch of DNA (<50 bp)
using a single PCR reaction. It requires two complementary 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 1015 bases at
both sides, which anneal to the template DNA. Following
cycles of DNA annealing and extension, the entire plasmid DNA containing the desired mutation is amplified
(Figure 2). Template DNA is selectively digested by
DpnI endonuclease to reduce background transformation. This procedure can also be executed without a
kit, using separate reagents, including polymerase, primers 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 simultaneously at multiple sites, the QuikChange Multi SiteDirected Mutagenesis Kit can be used [40]. It is based on
the same principle as the QuickChangeTM method, except 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 strategy 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].

Generation of mutation and deletion

constructs

If larger DNA fragments need to be inserted or replaced,

the restriction-free PCR-based cloning procedures described above can be applied (Figure 2). Deletions up
to 2.4 kb and insertions up to 3.5 kb have been successfully 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 mutations during the amplification of the DNA that could
affect protein expression.

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

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

Mutated
plasmid

Mutagenic
Primers

PCR

PCR

Vector
PCR

Template

New
clone

Mutated
plasmid
DpnI

DpnI

Template

Template

vector

PCR mega-primer

Ligase Cycling Reaction

Leftover
- strand
Cut
Vector

denature
anneal

Patched
+ strand

ligase

Ligated
+ strand

+
Insert 1 Insert 2

denature
anneal

Patched
new clone

ligase

Ligated
new
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

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.

Protein co-expression
A challenge in the field of structural biology is the production of protein complexes at amounts amenable for
functional and structural studies. Although in vitro reconstitution of such complexes can succeed, it is often a timeconsuming and complicated process that generally

Figure 3

Multiple vectors

Single vector
Multiple promoters

Single vector
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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Current Opinion in Structural Biology 2016, 38:145154

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 [4346].
Several comparative studies suggest that there is no preferred 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 control 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 reconstituted protein complex [44,47,48]. In any case, the selected cloning strategy to accomplish co-expression should
be flexible and parallelizable to allow a combinatorial
approach that is often crucial for the success of multiprotein expression. Until recently, classical restriction enzyme-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
Vector

Refs, Website

Fragment

Restriction enzyme/ligation-based cloning

Restriction
Restriction enzymes, T4 DNA Ligase
Golden Gate cloning
Type II endonuclease, T4 DNA Ligase
USER
USER enzyme mix

Restriction site
Restriction site
8-12 bp

Restriction site
Restriction site
dU in pimer

a,b,c,d,e

Ligation-based cloning
Topo1-TA

30 T-overhang

50 A-overhang

>12 bp LIC sites

No specific
No specific
No specific

>12 bp LIC sites

2040 bp
15 bp
1580 bp

[14]
[22]
c
[24]
a
[23]

No specific

15 bp

25 bp att site
34 bp loxP site
50 bp sequence,
I-SceI site
No specific

25 bp attB site
34 bp loxP site
50 bp sequence,
I-SceI site
1552 bp

No
No
No
No
No

1417 bp
2430 bp
30 bp
1525 bp
2025 bp

[32,41]
[33,34]
[36]
[37]
[38]

T4 DNA ligase

Exonuclease -dependent cloning/single strand DNA annealing

LIC
T4 DNA polymerase
SLIC
T4 DNA polymerase, RecA
In-Fusion1
InFusion HD cloning enzyme mix
11
Gibson assembly
T5 Exonuclease, Phusion DNA polymerase,
Taq ligase (Or proprietary enzyme mix)
Geneart1 seamless cloning
Proprietary enzyme mix
Recombination-dependent cloning
Gateway12
Proprietary Clonase enzyme mixtures
UPS
Cre Recombinase
MAGIC 3
I-Sce endonuclease, Reda Redb and Gam
(in vivo)
SLiCE 4
E. coli extract
PCR-based cloning/single-strand DNA annealing
PIPE
DNA polymerase
RF
DNA polymerase, DpnI
TPCR
DNA polymerase, DpnI
CPEC
DNA polymerase
EMP 5
DNA polymerase, T4 PNK, DpnI
T4 DNA Ligase
LCR 6
Thermostable ligase

specific
specific
specific
specific
specific

No specific

a
a

[26,27,42]
[25]
[28]
[30]

[39]

Remarks:
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/.
1

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Recombinant cloning strategies Celie, Parret and Perrakis 151

of poly-cistronic vectors for heterologous expression of

recombinant protein complexes [44,4951]. Generally, this
strategy implies that each coding sequence is cloned in a
donor plasmid flanked by unique restriction sites. Following 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 described 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.
Although originally tailored towards expression of eukaryotic 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 alternative approach amenable to multi-gene assembly is

based on yeast homologous recombination [57]. A variation of this method, the any-gene-any-plasmid (AGAP)
cloning, has recently been described and although not
widely applied, the authors highlight the general applicability of this method as a cost-effective efficient method
for multiprotein reconstitution in microbial and vertebrate 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 techniques have been developed in the field of synthetic
biology, such as Golden Braid for protein expression in
plants [59,60], USERec [61], unique nucleotide sequence (UNS)-guided assembly [62,63] and advanced
Gibson assembly methods [64]. LIC-based cloning methods are equally popular for multi-gene assembly of highly
repetitive genes for example in case of transcription
activator-like effector proteins [65].

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
Website

Tool
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

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/
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/
http://genomes.urv.es/OPTIMIZER/

Web portals
Portal for bioinformatic tools
Portal for bioinformatic tools
Human gene database

http://mobyle.pasteur.fr/cgi-bin/portal.py
http://www.expasy.org/
http://www.genecards.org/

Complete software packages

(freeware)
GenomeCompiler
SnapGene Viewer
SerialCloner
ApE

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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Reference

[73]

[74]
[75]

Current Opinion in Structural Biology 2016, 38:145154

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 comparison 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 fragments > 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 selection 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 topology. 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 recognition 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 availability of codon-optimized synthetic genes, expand the
versatility in protein expression methods and facilitate
screening of optimal protein production conditions.

References and recommended reading

Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1.

Smith HO, Wilcox KW: A restriction enzyme from Hemophilus

influenzae. I. Purification and general properties. J Mol Biol
1970, 51:379-391.

Current Opinion in Structural Biology 2016, 38:145154

2.

Yoshimori R, Roulland-Dussoix D, Boyer HW: R factor-controlled

restriction and modification of deoxyribonucleic acid:
restriction mutants. J Bacteriol 1972, 112:1275-1279.

3.

Cohen SN, Chang AC, Boyer HW, Helling RB: Construction of

biologically functional bacterial plasmids in vitro. Proc Natl
Acad Sci U S A 1973, 70:3240-3244.

4.

Chang AC, Cohen SN: Genome construction between bacterial

species in vitro: replication and expression of Staphylococcus
plasmid genes in Escherichia coli. Proc Natl Acad Sci U S A
1974, 71:1030-1034.

5.

Morrow JF, Cohen SN, Chang AC, Boyer HW, Goodman HM,
Helling RB: Replication and transcription of eukaryotic DNA in
Escherichia coli. Proc Natl Acad Sci U S A 1974, 71:1743-1747.

6.

Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT,
Mullis KB, Erlich HA: Primer-directed enzymatic amplification
of DNA with a thermostable DNA polymerase. Science 1988,
239:487-491.

7.

Hughes RA, Miklos AE, Ellington AD: Gene synthesis: methods

and applications. Methods Enzymol 2011, 498:277-309.

8.

Baumann T, Arndt KM, Muller KM: Directional cloning of DNA

fragments using deoxyinosine-containing oligonucleotides
and endonuclease V. BMC Biotechnol 2013, 13:81.

9.

Padgett KA, Sorge JA: Creating seamless junctions

independent of restriction sites in PCR cloning. Gene 1996,
168:31-35.

10. Engler C, Kandzia R, Marillonnet S: A one pot, one step,

precision cloning method with high throughput capability.
PLoS ONE 2008, 3:e3647.
11. Engler C, Gruetzner R, Kandzia R, Marillonnet S: Golden gate
shuffling: a one-pot DNA shuffling method based on type IIs
restriction enzymes. PLoS ONE 2009, 4:e5553.
12. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S: A
modular cloning system for standardized assembly of
multigene constructs. PLoS ONE 2011, 6:e16765.
13. Bitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R,
Vaiskunaite R: USER friendly DNA engineering and cloning
method by uracil excision. Nucleic Acids Res 2007,
35:1992-2002.
14. Aslanidis C, de Jong PJ: Ligation-independent cloning of PCR
products (LIC-PCR). Nucleic Acids Res 1990, 18:6069-6074.
15. Shuldiner AR, Scott LA, Roth J: PCR-induced (ligase-free)
subcloning: a rapid reliable method to subclone polymerase
chain reaction (PCR) products. Nucleic Acids Res 1990,
18:1920.
16. Hsiao K: Exonuclease III induced ligase-free directional
subcloning of PCR products. Nucleic Acids Res 1993,
21:5528-5529.
17. Weeks SD, Drinker M, Loll PJ: Ligation independent cloning
vectors for expression of SUMO fusions. Protein Expr Purif
2007, 53:40-50.
18. Eschenfeldt WH, Maltseva N, Stols L, Donnelly MI, Gu M, Nocek B,
Tan K, Kim Y, Joachimiak A: Cleavable C-terminal His-tag
vectors for structure determination. J Struct Funct Genomics
2010, 11:31-39.
19. Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, BurgessBrown NA, Gileadi O: High-throughput production of human
proteins for crystallization: the SGC experience. J Struct Biol
2010, 172:3-13.
20. Luna-Vargas MP, Christodoulou E, Alfieri A, van Dijk WJ,
Stadnik M, Hibbert RG, Sahtoe DD, Clerici M, Marco VD, Littler D
et al.: Enabling high-throughput ligation-independent cloning
and protein expression for the family of ubiquitin specific
proteases. J Struct Biol 2011, 175:113-119.
21. Thieme F, Engler C, Kandzia R, Marillonnet S: Quick and clean
cloning: a ligation-independent cloning strategy for selective
cloning of specific PCR products from non-specific mixes.
PLoS ONE 2011, 6:e20556.
www.sciencedirect.com

Recombinant cloning strategies Celie, Parret and Perrakis 153

22. Li MZ, Elledge SJ: Harnessing homologous recombination in

vitro to generate recombinant DNA via SLIC. Nat Methods 2007,
4:251-256.
23. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd,
Smith HO: Enzymatic assembly of DNA molecules up to several
hundred kilobases. Nat Methods 2009, 6:343-345.
24. Benoit RM, Wilhelm RN, Scherer-Becker D, Ostermeier C: An
improved method for fast, robust, and seamless integration of
DNA fragments into multiple plasmids. Protein Expr Purif 2006,
45:66-71.
25. Liu Q, Li MZ, Leibham D, Cortez D, Elledge SJ: The univector
plasmid-fusion system, a method for rapid construction of
recombinant DNA without restriction enzymes. Curr Biol 1998,
8:1300-1309.
26. Hartley JL, Temple GF, Brasch MA: DNA cloning using in vitro
site-specific recombination. Genome Res 2000, 10:1788-1795.
27. Liang X, Peng L, Baek CH, Katzen F: Single step BP/LR
combined Gateway reactions. Biotechniques 2013, 55:265-268.
28. Li MZ, Elledge SJ: MAGIC, an in vivo genetic method for the
rapid construction of recombinant DNA molecules. Nat Genet
2005, 37:311-319.
29. Busso D, Delagoutte-Busso B, Moras D: Construction of a set
Gateway-based destination vectors for high-throughput
cloning and expression screening in Escherichia coli. Anal
Biochem 2005, 343:313-321.
30. Zhang Y, Werling U, Edelmann W: SLiCE: a novel bacterial cell
extract-based DNA cloning method. Nucleic Acids Res 2012,
40:e55.

41. Klock HE, Lesley SA: The Polymerase Incomplete Primer

Extension (PIPE) method applied to high-throughput cloning
and site-directed mutagenesis. Methods Mol Biol 2009,
498:91-103.
42. Liang X, Peng L, Li K, Peterson T, Katzen F: A method for multisite-directed mutagenesis based on homologous
recombination. Anal Biochem 2012, 427:99-101.
43. Selleck W, Tan S: Recombinant protein complex expression in
E. coli. Curr Protoc Protein Sci 2008, 5:21 (Chapter 5:Unit).
44. Romier C, Ben Jelloul M, Albeck S, Buchwald G, Busso D,
Celie PH, Christodoulou E, De Marco V, van Gerwen S,
Knipscheer P et al.: Co-expression of protein complexes in
prokaryotic and eukaryotic hosts: experimental procedures,
database tracking and case studies. Acta Crystallogr D Biol
Crystallogr 2006, 62:1232-1242.
45. Busso D, Peleg Y, Heidebrecht T, Romier C, Jacobovitch Y,
Dantes A, Salim L, Troesch E, Schuetz A, Heinemann U et al.:
Expression of protein complexes using multiple Escherichia
coli protein co-expression systems: a benchmarking study. J
Struct Biol 2011, 175:159-170.
46. Kerrigan JJ, Xie Q, Ames RS, Lu Q: Production of protein
complexes via co-expression. Protein Expr Purif 2011, 75:1-14.
47. Diebold ML, Fribourg S, Koch M, Metzger T, Romier C:
Deciphering correct strategies for multiprotein complex
assembly by co-expression: application to complexes as large
as the histone octamer. J Struct Biol 2011, 175:178-188.
48. Johnston K, Clements A, Venkataramani RN, Trievel RC,
Marmorstein R: Coexpression of proteins in bacteria using T7based expression plasmids: expression of heteromeric cellcycle and transcriptional regulatory complexes. Protein Expr
Purif 2000, 20:435-443.

31. Zhang Y, Werling U, Edelmann W: Seamless Ligation Cloning

 Extract (SLiCE) cloning method. Methods Mol Biol 2014,
1116:235-244.
Demonstration of SLiCE cloning method, that uses bacterial extracts as
the source of enzymes for molecular cloning, providing a cheap method
for versatile cloning.

49. Tan S, Kern RC, Selleck W: The pST44 polycistronic expression

system for producing protein complexes in Escherichia coli.
Protein Expr Purif 2005, 40:385-395.

32. Klock HE, Koesema EJ, Knuth MW, Lesley SA: Combining the
polymerase incomplete primer extension method for cloning
and mutagenesis with microscreening to accelerate structural
genomics efforts. Proteins 2008, 71:982-994.

50. Anderson M, Huh JH, Ngo T, Lee A, Hernandez G, Pang J,

Perkins J, Dutnall RN: Co-expression as a convenient method
for the production and purification of core histones in
bacteria. Protein Expr Purif 2010, 72:194-204.

33. Unger T, Jacobovitch Y, Dantes A, Bernheim R, Peleg Y:

Applications of the Restriction Free (RF) cloning procedure for
molecular manipulations and protein expression. J Struct Biol
2010, 172:34-44.

51. Takai K, Hisamatsu K: SfiNX: a method for assembly of protein

coding sequences with high success rates. Biotechnol Lett
2016, 38:773-778.

34. van den Ent F, Lowe J: RF cloning: a restriction-free method for

inserting target genes into plasmids. J Biochem Biophys
Methods 2006, 67:67-74.
35. Bryksin AV, Matsumura I: Overlap extension PCR cloning: a
simple and reliable way to create recombinant plasmids.
Biotechniques 2010, 48:463-465.
36. Erijman A, Dantes A, Bernheim R, Shifman JM, Peleg Y: TransferPCR (TPCR): a highway for DNA cloning and protein
engineering. J Struct Biol 2011, 175:171-177.
37. Quan J, Tian J: Circular polymerase extension cloning of
complex gene libraries and pathways. PLoS ONE 2009,
4:e6441.
38. Ulrich A, Andersen KR, Schwartz TU: Exponential megapriming
PCR (EMP) cloningseamless DNA insertion into any target
plasmid without sequence constraints. PLoS ONE 2012,
7:e53360.
39. de Kok S, Stanton LH, Slaby T, Durot M, Holmes VF, Patel KG,

Platt D, Shapland EB, Serber Z, Dean J et al.: Rapid and reliable
DNA assembly via ligase cycling reaction. ACS Synth Biol 2014,
3:97-106.
Counterpart of Polymerase Chain Reaction (PCR). Instead of a polymerase, a thermostable ligase is used to fuse DNA fragments during denaturation/annealing/ligation cycles.
40. Hogrefe HH, Cline J, Youngblood GL, Allen RM: Creating
randomized amino acid libraries with the QuikChange
Multi Site-Directed Mutagenesis Kit. Biotechniques 2002, 33
11581160, 1162, 11641155.
www.sciencedirect.com

52. Casini A, MacDonald JT, De Jonghe J, Christodoulou G,

Freemont PS, Baldwin GS, Ellis T: One-pot DNA: construction for
synthetic biology: the Modular Overlap-Directed Assembly
with Linkers (MODAL) strategy. Nucleic Acids Res 2014, 42:e7.
53. Casini A, Storch M, Baldwin GS, Ellis T: Bricks and blueprints:
 methods and standards for DNA assembly. Nat Rev Mol Cell
Biol 2015, 16:568-576.
Comprehensive review about cloning methods for protein expression
with excellent illustrations to highlight the cloning principles of each
system.
54. Berger I, Fitzgerald DJ, Richmond TJ: Baculovirus expression
system for heterologous multiprotein complexes. Nat
Biotechnol 2004, 22:1583-1587.
55. Bieniossek C, Imasaki T, Takagi Y, Berger I: MultiBac: expanding
the research toolbox for multiprotein complexes. Trends
Biochem Sci 2012, 37:49-57.
56. Zhang Z, Yang J, Barford D: Recombinant expression and

reconstitution of multiprotein complexes by the USER cloning
method in the insect cell-baculovirus expression system.
Methods 2016, 95:13-25.
Application of USER cloning strategy to generate multigene containing
baculovirus transfer vectors for insect cell co-expression, based on the
MultiBac approach.
57. Oldenburg KR, Vo KT, Michaelis S, Paddon C: Recombinationmediated PCR-directed plasmid construction in vivo in yeast.
Nucleic Acids Res 1997, 25:451-452.
58. Joska TM, Mashruwala A, Boyd JM, Belden WJ: A universal
cloning method based on yeast homologous recombination
Current Opinion in Structural Biology 2016, 38:145154

154 New constructs and expression of proteins

that is simple, efficient, and versatile. J Microbiol Methods 2014,

100:46-51.
59. Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P,
Fernandez-del-Carmen A, Granell A, Orzaez D: GoldenBraid: an
iterative cloning system for standardized assembly of
reusable genetic modules. PLoS ONE 2011, 6:e21622.
60. Sarrion-Perdigones A, Palaci J, Granell A, Orzaez D: Design and

construction of multigenic constructs for plant biotechnology
using the GoldenBraid cloning strategy. Methods Mol Biol 2014,
1116:133-151.
This paper describes the GoldenBraid cloning system that allows assembly of multi-gene constructs for protein expression in plants.
61. Villiers B, Hollfelder F: USER friendly DNA recombination
(USERec): gene library construction requiring minimal
sequence homology. Methods Mol Biol 2014, 1179:213-224.
62. Torella JP, Boehm CR, Lienert F, Chen JH, Way JC, Silver PA:
Rapid construction of insulated genetic circuits via synthetic
sequence-guided isothermal assembly. Nucleic Acids Res
2014, 42:681-689.
63. Torella JP, Lienert F, Boehm CR, Chen JH, Way JC, Silver PA:
Unique nucleotide sequence-guided assembly of repetitive
DNA parts for synthetic biology applications. Nat Protoc 2014,
9:2075-2089.

67. Rebatchouk D, Daraselia N, Narita JO: NOMAD: a versatile

strategy for in vitro DNA manipulation applied to promoter
analysis and vector design. Proc Natl Acad Sci U S A 1996,
93:10891-10896.
68. Shetty RP, Endy D, Knight TF Jr: Engineering BioBrick vectors
from BioBrick parts. J Biol Eng 2008, 2:5.
69. Rokke G, Korvald E, Pahr J, Oyas O, Lale R: BioBrick assembly
standards and techniques and associated software tools.
Methods Mol Biol 2014, 1116:1-24.
70. Mooij WT, Mitsiki E, Perrakis A: ProteinCCD: enabling the design
of protein truncation constructs for expression and
crystallization experiments. Nucleic Acids Res 2009,
37:W402-W405.
71. Salis HM, Mirsky EA, Voigt CA: Automated design of synthetic
ribosome binding sites to control protein expression. Nat
Biotechnol 2009, 27:946-950.
72. Espah Borujeni A, Channarasappa AS, Salis HM: Translation rate

is controlled by coupled trade-offs between site accessibility,
selective RNA unfolding and sliding at upstream standby sites.
Nucleic Acids Res 2014, 42:2646-2659.
Computational modeling and experimental validation of translation initiation parameters that have been added to the RBS calculator available at
http://salis.psu.edu/software for design and optimization of protein
expression constructs.

64. Akama-Garren EH, Joshi NS, Tammela T, Chang GP, Wagner BL,
Lee DY, Rideout Iii WM, Papagiannakopoulos T, Xue W, Jacks T: A
modular assembly platform for rapid generation of DNA
constructs. Sci Rep 2016, 6:16836.

73. Dong X, Stothard P, Forsythe IJ, Wishart DS: PlasMapper: a web

server for drawing and auto-annotating plasmid maps. Nucleic
Acids Res 2004, 32:W660-W664.

65. Schmid-Burgk JL, Schmidt T, Hornung V: Ligation-independent

cloning (LIC) assembly of TALEN genes. Methods Mol Biol 2015,
1239:161-169.

74. Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC,

Jahn D: JCat: a novel tool to adapt codon usage of a target
gene to its potential expression host. Nucleic Acids Res 2005,
33 W526531.

66. Stevenson J, Krycer JR, Phan L, Brown AJ: A practical

comparison of ligation-independent cloning techniques. PLOS
ONE 2013, 8:e83888.

75. Puigbo P, Guzman E, Romeu A, Garcia-Vallve S: OPTIMIZER: a

web server for optimizing the codon usage of DNA sequences.
Nucleic Acids Res 2007, 35:W126-W131.

Current Opinion in Structural Biology 2016, 38:145154

www.sciencedirect.com