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Critical reflections on synthetic gene design for
recombinant protein expression
1 2 1
Annabel HA Parret , Hu¨ seyin Besir and Rob Meijers
Gene synthesis enables the exploitation of the degeneracy of expression using large-scale fermentation [2]. However,
the genetic code to boost expression of recombinant protein large-scale fermentation often requires optimization to
targets for structural studies. This has created new address problems of plasmid loss, oxygen deprivation and
opportunities to obtain structural information on proteins that pH fluctuation [3].
are normally present in low abundance. Unfortunately,
synthetic gene expression occasionally leads to insoluble or A more cost-effective approach is the optimization of the
misfolded proteins. This could be remedied by recent insights protein source, either by opting for a protein species of
in the effect of codon usage on translation initiation and high natural abundance, or by using an efficient recom-
elongation. In this review, we discuss the interplay between binant expression system. Traditionally, selecting protein
optimal gene and vector design to enhance expression in a homologues from species that can be purified from natural
particular host and highlight the benefits and potential pitfalls source has been rather successful [4–8]. Nevertheless,
associated with protein expression from synthetic genes. there is an increasing need to obtain the structure of a
protein from a particular species, in order to assess spe-
Addresses
1 cies-specific functional aspects. Structures of human or
European Molecular Biology Laboratory, Hamburg Unit, Notkestrasse
85, 22607 Hamburg, Germany murine proteins involved in the immune or nervous
2
Protein Expression and Purification Core Facility, European Molecular system can help to relate the effect of specific point
Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
mutants or posttranslational modifications. Similarly,
structural analysis of virulent proteins from bacterial
Corresponding author: Meijers, Rob ([email protected])
pathogens can reveal species-specific details important
for drug design. In this review, we focus on the oppor-
Current Opinion in Structural Biology 2016, 38:155–162 tunities that gene synthesis provides to optimize recom-
This review comes from a themed issue on New constructs and binant protein expression of specific protein targets.
expression of proteins
Edited by Rob Meijers and Anastassis Perrakis
Application of synthetic genes for protein
For a complete overview see the Issue and the Editorial production
Available online 21st July 2016 The use of synthetic genes for the production of proteins
http://dx.doi.org/10.1016/j.sbi.2016.07.004 for structural characterization is widespread. In general,
gene synthesis may expedite the production of a series of
0959-440X/# 2016 The Authors. Published by Elsevier Ltd. This is an
constructs with engineered restriction sites. It is often
open access article under the CC BY-NC-ND license (http://creative-
commons.org/licenses/by-nc-nd/4.0/). preferred to the use of cDNA, which can contain several
isoforms or splice variants. For target proteins identified
from metagenomes or extremophiles, gene synthesis may
provide the only route to protein expression, because
cDNA is not available. Protein expression from codon-
optimized genes has been especially successful for pro-
Introduction teins involved in cell signalling whose native codon
The efficient overexpression of proteins is a prerequisite sequence can be strongly regulated, such as Irisin [9],
for successful structure determination. Structural geno- Parkin [10] and Netrin [11]. Codon optimization has also
mics studies on a wide range of soluble protein targets uncovered hidden levels of gene regulation that are
over more than a decade have shown that more than 40% encoded in codon bias. Codon optimization of the
of the constructs failed due to limited expression FRQ protein involved in the regulation of circadian
(Figure 1). The high failure rate is a result of the use rhythms resulted in a loss of function, although expres-
of standardized approaches, and can serve as a baseline for sion levels were increased [12 ]. Codon optimization also
a structure determination project on a specific target. To revealed a hidden quality control mechanism encoded in
alleviate these ominous statistics, a number of approaches the gene of the cystic fibrosis transmembrane conduc-
have been adopted which address recombinant protein tance regulator (CFTR), an anion channel membrane
expression. The use of parallel cloning strategies employ- protein [13]. Similarly, optimization of the CTP1L endo-
ing different construct lengths and purification tags can lysin gene led to the discovery of a secondary translation
increase success rate to a certain extent [1]. Alternatively, site that produced a truncated protein. The truncated
larger amounts of protein can be obtained by scaling up protein forms a 1:1 complex with the full length protein,
www.sciencedirect.com Current Opinion in Structural Biology 2016, 38:155–162
156 New constructs and expression of proteins
Figure 1
ed features that have been identified to hamper expres-
sion (Table 2). Gene design tools enable researchers to
40000 optimize the sequence of their gene of interest (GOI)
2004 [20]. A common strategy to manipulate codon bias to
35000 100%
100% 2016 maximize protein translation is based on the ‘codon
30000 adaptation index’ (CAI) [21]. CAI is a species-specific
index for codon frequency based on a set of highly
25000
expressing genes. Individual genes can then be analyzed,
60%
20000 comparing the actual codons used to a fully optimized
51% gene that contains only the most frequently used codons.
15000
It should be noted that CAI maximization does not
No. of targets
10000 necessarily correlate with high protein yield [22–25]. In
9% 23%
1 general, the best results using CAI optimization are 17%
5000
* obtained when a subset of less frequent codons is
4% 3% 3%
0 replaced, after which a secondary list of criteria should
Cloned Expressed Purified Experiment PDB deposit be optimized involving DNA and RNA sequence ele-
Milestones ments that can negatively influence expression of the
Current Opinion in Structural Biology GOI (Table 2). It appears some features of codon bias are
universal for all expression systems, whereas others are
specific for prokaryotic or eukaryotic expression.
A comparison of the total number of targets achieved per milestone
along a structure genomics pipeline as reported by TargetDB in
2004 [104] and as of 15th of March 2016 (http://targetdb.rcsb.org/
Codon optimization algorithms will generally allow ad-
metrics/). The recent data are compiled from soluble protein targets
justment of several of these undesirable sequence prop-
from three consortia (JCSG, NESG and NYSGRC). The attrition rate
erties simultaneously [20,26–28], although the weight
from clone to purified target has changed little over a decade and
hovers around 75%, indicating that the early stages of a structure given to each sequence parameter varies between differ-
determination project are most crucial for successful completion. *The ent algorithms. It is important to note that proprietary
definition of the milestone ‘Experiment’ has changed from ‘crystals’ to
CAI-based codon optimization algorithms adjust a subset
‘X-ray/NMR/EM studies performed for structural studies’. As a result,
of rare codons in a random manner. As a result, gene
there is a large discrepancy between crystals obtained (4% in 2004)
optimization is ambiguous, since the replacement of rare
and experiments done on the sample (17%), irrespective of the
outcome. codons is not based on empirical data. Species-specific
gene optimization routinely involves the use of the spe-
cies-relevant CAI index, taking into account the codon
and lysis activity is substantially reduced when the sec-
frequency of the particular species. Other factors that
ondary translation site is removed by codon optimization
affect for example eukaryotic protein expression are most
[14]. Toll-like receptors (TLRs) contain leucine-rich
often not considered, because the influence on expression
domains, and it was shown through targeted leucine
levels is poorly understood.
codon optimization that transcription of TLRs is regulat-
ed by leucine codons to balance the abundance of certain
Recent insights into the relationship between
receptors [15]. It is clear that gene synthesis may provide
mRNA, protein expression and protein folding
even unexpected benefits, but there are also numerous
It is obvious that codon-mediated translational control is
reports were it leads to reduced protein yield [16,17].
not yet well understood, and further insight will benefit
gene design for protein expression. Recent studies indicate
Adapting codon usage of target genes using that rare codons may play different roles, such as safe-
codon optimization algorithms guarding mRNA structure [29], depending on their posi-
Exploiting gene synthesis for transgene expression tioning within the gene. A large scale study on bacterial
enables researchers to influence the multitude of vari- genes expression in Escherichia coli under a common pro-
ables controlling protein yield. The biased usage of motor system revealed that mRNA stability correlates with
synonymous codons has a strong effect on protein expression levels [30 ]. However, there is a difference in
expression [18 ]. Species-specific differences in this the effect between the ‘head’ of the gene (covering ap-
so-called codon bias can result in tRNA depletion, proximately 16 codons) and the rest of the gene. In the
which slows down protein translation. This effect can ‘head’, the mRNA structure is more important, whereas the
be in part compensated by using an expression host rest of the gene is rather affected by codon-mediated
that expresses rare tRNAs [16], however this is often translation elongation efficiency. This phenomenon is
insufficient [19]. likely a universal property for both prokaryotic and eukary-
otic protein expression [31]. Cell-free expression studies
An extensive collection of software tools (Table 1) have shown that universal translation initation tags can be
are available to analyze codon usage and identify unwant- introduced upstream of the GOI that boost expression both
Current Opinion in Structural Biology 2016, 38:155–162 www.sciencedirect.com
The use of synthetic genes for protein expression Parret, Besir and Meijers 157
Table 1
Various tools to evaluate and optimize DNA sequences
Application Name Web URL Reference
Codon usage database http://www.kazusa.or.jp/codon/ [68]
Codon bias database CBDB http://homepages.luc.edu/ cputonti/cbdb/ [69]
Analysis of codon usage RaCC http://nihserver.mbi.ucla.edu/RACC/
CAIcaI http://genomes.urv.cat/CAIcal/ [70]
CAI calculator http://www.umbc.edu/codon/cai/cais.php [71]
CodonO http://sysbio.cvm.msstate.edu/CodonO/ [72]
codonW http://codonw.sourceforge.net/
GCUA http://gcua.schoedl.de/ [73]
INCA http://bioinfo.hr/research/inca/ [74]
http://www.codons.org/index.html [38] http://www.cmbl.uga.edu/software/codon_usage.html
http://www.bioinformatics.org/sms2/codon_usage.html [75]
a b
Gene design tools CodonOpt https://eu.idtdna.com/CodonOpt b
COOL http://cool.syncti.org/ [76] b
DNAWorks https://hpcwebapps.cit.nih.gov/dnaworks/ [77] c
D-Tailor https://sourceforge.net/projects/dtailor/ [78] c
EuGene http://bioinformatics.ua.pt/eugene/ [79] b
GeneDesign http://www.genedesign.org [80]
c
Gene Designer https://www.dna20.com/resources/genedesigner [81] GeneOptimizerb https://www.thermofisher.com/de/de/home/life- [82] science/cloning/gene-synthesis/geneart-gene- synthesis/geneoptimizer.html b
JCat http://www.jcat.de/ [83]
c
mRNA Optimizer http://bioinformatics.ua.pt/software/mRNA-optimiser [84] b
OPTIMIZER http://genomes.urv.es/OPTIMIZER/ [85]
OptimumGeneb http://www.genscript.com/cgi-bin/tools/rare_codon_analysis
Synthetic Gene http://userpages.umbc.edu/ wug1/codon/sgd/ [86]
Designerb
Visual Gene http://visualgenedeveloper.net/index.html [87] Developerc
a
A very useful and comprehensive comparison of functionality and easiness of use of gene design tools for codon optimization is described by Gould
et al. [26]. b
Web-based tool. c
Stand-alone software.
in bacterial and eukaryotic systems [32,33]. To boost Bioinformatic analyses indicate that rare codon clusters
0
protein expression, it may be sufficient to modify the codon are found in a wide variety of genes, especially in the 5
0
usage at the ‘head’ of the gene in order to optimize the and 3 termini [38–40]. Such clusters are thought to
mRNA structure for efficient translation initiation, and slowdown the ribosome during translation elongation in
leave the codon sequence of the rest of the gene intact. order to allow co-translational protein folding [41–43].
Slow elongation is especially important in case of mem-
In prokaryotes, translation of a transcript is initiated before brane protein targeting [44 ,45,46]. As a consequence,
the transcription process is complete, due to the recruit- modifying the codons involved in the regulation of trans-
ment of ribosomes to the newly synthesized mRNA. In lation speed can culminate in protein misfolding. In one
genes which are not highly expressed, the translation particular case, the replacement of frequently used
efficiency is not necessarily correlated with protein syn- codons by rare codons at domain boundaries resulted
thesis rates, as ribosome profiling studies have shown [34– in an improved solubility of an enzyme produced in E.
36]. These studies point to another level of regulation, coli due to slower translation rate at these sites [47]. To
where codon usage affects protein folding and post-trans- circumvent deleterious effects of codon usage during co-
lational modifications. A bioinformatics analysis in E. coli translational protein folding, Angov et al. proposed a so-
revealed that putative translational attenuation sites are called codon harmonization strategy where rare codons
widespread, and were identified in about 60% of protein are positioned where domain boundaries were predicted
coding sequences [37]. In this study, Li et al. proposed that [48,49]. When compared to conventional codon optimi-
these internal Shine–Dalgarno sequences are the main zation, this strategy resulted in improved expression level
determinants of translation elongation rates in bacteria and soluble protein yield when overexpressing optimized
allowing for translational pausing where necessary. genes from the eukaryotic parasite Plasmodium falciparum
www.sciencedirect.com Current Opinion in Structural Biology 2016, 38:155–162
158 New constructs and expression of proteins
Table 2
Codon sequence features potentially influencing protein expression levels
Sequence feature Possible effect References
General features
G + C content [15,22,88,89]
Very high G + C content (>70%) mRNA secondary structure formation; can slow down or inhibit translation
Very low G + C content (<30%) Can slow down transcription elongation
mRNA secondary structure [31,53,56 ,90–92]
Global Can promote mRNA stability
Near RBS Reduction of translational initiation
Repetitive sequences Inhibition of translation through formation of mRNA stem–loops [16]
Rare codons
Global Can result in translational pausing, tRNA depletion resulting in low [27,39,93,94]
protein yield, mRNA degradation
At protein domain boundaries Promotes accurate co-translational folding [40–42,48]
Alternative start codons Shorter protein (if in frame); mixture of two proteins; [14]
incorrect protein if out of frame
Bacterial features
Chi-site stretches Recombination hotspots; DNA instability [95]
Alternative transcriptional initiation sites Leaderless transcripts; shorter or incorrect protein [96–98]
RNase E sites Can effect mRNA stability [31,99]
SD-like RBS sequences Can cause translational pausing (ribosome stalling) [37]
Eukaryotic features
High CpG content Can result in incorrect transcription initiation [88,100]
Internal TATA-boxes Can result in incorrect transcription initiation
Cryptic splice sites Missplicing of mRNA; incorrect protein [101]
Potential polyA sites Can induce premature termination of translation [102,103]
in E. coli. All these studies indicate that in gene synthesis, recombinant protein expression involves three intersect-
it may be important to conserve codon usage in some parts ing areas. First, the choice of expression host, second, the
of the gene, to maintain the regulatory elements that selection of the expression vector [50] and third, the GOI
affect folding and post-translational modifications. (Figure 2). Much research has been done to optimize
expression vectors adapted to the expression host. How-
The interface between the synthetic gene and ever, the adaptation of the GOI towards the expression
the expression vector vector is often neglected. Most bacterial vector systems
It is important to keep in mind that the mRNA produced are used as plug-and-play, where the GOI is inserted
to translate into protein is often not limited to the downstream of a vector-encoded ribosome binding site
0 0
synthetized gene, but contains fragments derived from (RBS). As part of the 5 untranslated region (5 UTR), the
the expression vector. In fact, the challenge to optimize RBS controls the rate of ribosome recruitment as well as
Figure 2
GOI vector insert
RNA pool
Protein start of transcript yield RBS translationalaffinity start tag protease GOIsite
AUG * 5’ UTR Host Vector 10 bp protein synthesis integration
Current Opinion in Structural Biology
Optimization triangle illustrating the key determinants in recombinant protein expression. The inset illustrates the anatomy of a typical bacterial
overexpression vector belonging to the popular pET-series in the region surrounding the insertion site of the GOI. The asterisk indicates a potential
scar resulting from the cloning strategy.
Current Opinion in Structural Biology 2016, 38:155–162 www.sciencedirect.com
The use of synthetic genes for protein expression Parret, Besir and Meijers 159
the efficiency of translation initiation [51]. It is generally improve recombinant protein expression. Hence, spe-
0
assumed that the mRNA sequence of the 5 UTR of an cies-specific gene optimization using proprietary algo-
expression vector is optimized in order to maximize the rithms can increase protein yield. Unfortunately,
translational efficiency of the GOI within a particular predicting the output of the different optimization algo-
host. However, the mRNA structure of the initial 40 rithms is still a major challenge, especially for membrane
coding nucleotides can be crucial to obtain high protein and secreted proteins, due to the many parameters influ-
expression levels, given that in general strong secondary encing codon usage. It would be a great advance if
structure (due to e.g. high GC content) inhibits efficient commonly used codon optimization algorithms were im-
translation initiation [22,24,52–55,56 ,57]. An additional proved in the future to incorporate the new insights
global observation is the relatively higher abundance of obtained in recent years with respect to codon usage.
rare codons in the initial 30–50 codons [58]. The corre- This will probably involve segmentation of the gene,
lation between the presence of rare codons and weak where each segment has different requirements for codon
0
mRNA structure in the 5 end of coding sequences is still optimization, including segments for translation initia-
under debate [55]. tion, domain boundaries and sites for post-translational modifications.
0
In typical bacterial vectors, the 5 end of the coding
sequence (excluding the GOI) often encodes secretion Optimized protein translation initiation could be
signals, affinity tags, fusion proteins or linker remnants addressed by vector design, rather than codon optimiza-
resulting from cloning, but the mRNA structure in this tion of the gene of interest. Indeed, synonymous ex-
region is rarely scrutinized (Figure 2). This may explain change of a small number of codons or introducing
0
why the expression of some proteins is strongly affected non-coding secondary structure elements at the 5 -end
by the placement of a tag on either the N-terminus or C- of a gene can already be sufficient for a significant increase
terminus or by the length and sequence of the linker in expression levels [65,66]. This can be combined with
region between tag and first codon of the transgene. N- gene fragment synthesis, consisting of linear DNA frag-
terminal tags may have evolved towards optimal mRNA ments that can be used for direct cloning into expression
structure, thus increasing expression of some proteins vectors or as template for PCR amplification. In this
with a peculiar codon sequence at the start of the gene. respect, the Biobricks movement (http://biobricks.org/)
For example, it has been shown that addition of a leader is a promising initiative to deliver direct access to tools to
sequence can overcome the notoriously low expression of make proteins from genetically optimized building blocks
AT-rich genes in E. coli [59]. On the basis of the recent [67].
evidence on the importance of codon usage at the trans-
lation initiation site, the choice of any expression vector The expectation is that broad studies on the relationship
should include a review of this region. If the protein between codon usage and tRNA abundance as well as
expression is suboptimal, it could be worthwhile to opti- mRNA structure and stability will lead to a set of robust
mize elements involved in translation initiation upstream rules for gene design. This will further enable efficient
of the GOI and include these in gene synthesis. protein expression that is affordable for the larger molec-
ular biology community.
In eukaryotes, translation initiation is an intricate stepwise
process orchestrated by multiple components. Extensive Conflict of interest statement
studies in Saccharomyces cerevisiae in particular revealed that Nothing declared.
key regulatory elements, i.e. translation initiation motifs
0
and secondary structures in the 5 -UTR, modulate protein Acknowledgements
abundance [58,60–62]. One particularly successful ap- The authors are very grateful to Xia Sheng (Genscript) and Michel Koch for
providing valuable input into the manuscript. We would like to apologize to
proach to manipulate protein yield in mammalian cells
those authors whose work could not be cited here due to space restrictions.
has been the incorporation of viral introns and optimized
Kozak sequences into the translation initiation sequence of
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