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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)
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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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
attP1
ccdB - lethal gene
Donor
Vector
attP2
BP
Destination
Vector
clo
n
as
attB1
E. coli
attR2
attB1
LR clonase
attL1
Expression
Clone
Transformation
attB2
attB2
PCR product or
vector containing gene
Entry
Clone
attL2
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.
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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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
Current Opinion in Structural Biology 2016, 38:145154
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Figure 2
Quickchange mutagenesis
Mutated
plasmid
Mutagenic
Primers
PCR
PCR
Vector
PCR
Template
New
clone
Mutated
plasmid
DpnI
DpnI
Template
Template
vector
PCR mega-primer
denature
anneal
Patched
+ strand
ligase
Ligated
+ strand
+
Insert 1 Insert 2
denature
anneal
Patched
new clone
ligase
Ligated
new
clone
+
Insert 1
Insert 2
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.
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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Table 1
Cloning methods for creating protein expression constructs
Cloning strategy
Enzymes required
Cloning site/sequence
Vector
Refs, Website
Fragment
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
[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
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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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].
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/
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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