Biotechnol Lett (2013) 35:1971–1981 DOI 10.1007/s10529-013-1396-y

REVIEW

Expanding the landscape of recombinant protein production in Escherichia coli Alejandro Hochkoeppler

Received: 19 April 2013 / Accepted: 26 June 2013 / Published online: 30 October 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Over the years, several vectors and host strains have been constructed to improve the overexpression of recombinant proteins in Escherichia coli. More recently, attention has focused on the coexpression of genes in E. coli, either by means of a single vector or by cotransformation with multiple compatible plasmids. Co-expression was initially designed to generate protein complexes in vivo, and later served to extend the use of E. coli as a platform for the production of heterologous proteins. This review shows how the co-expression of genes in E. coli is challenging the production of protein complexes and proteins bearing post-translational modifications or unnatural amino acids. In addition, the importance of co-expression to achieve efficient secretion of recombinant proteins in E. coli is discussed, with recent insights into the use of coexpression to overproduce membrane proteins. Keywords Co-expression  Compatible plasmids  Escherichia coli  Post-translational modifications  Protein complexes  Protein production  Unnatural amino acids A. Hochkoeppler (&) Department of Pharmacy and Biotechnology, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy e-mail: [email protected] A. Hochkoeppler CSGI, University of Florence, Via Della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy

Introduction Modern biologists are deeply indebted to Escherichia coli. This profitable prokaryote provided the lac and araBAD operons (Mu¨ller-Hill 1996; Schleif 2000), two incomparable case studies to dissect gene expression into arrays of regulatory elements. Indeed, promoters, operators, transcriptional and translational regulators were first identified using E. coli as a model organism. Later on, a vast repertoire of well-characterized E. coli gene expression regulators was introduced into biotechnology. In particular, gene overexpression systems started to be designed and tested leading to the industrial production of valuable proteins like insulin (Tibaldi 2012) and interferons (Langer and Pestka 1984; Baron and Narula 1990). More recently, a number of sophisticated plasmids for protein overexpression in E. coli have been reported (for a recent review see Durani et al. 2012), and appropriate host strains genetically isolated. These new tools were devised to improve gene overexpression according to different strategies: (i) the development of tightly regulated expression vectors (Guzman et al. 1995; Mertens et al. 1995) suitable for the production of toxic proteins such as those whose expression leads to host death (Miroux and Walker 1996); (ii) the use of high- or low-copy number plasmids (Table 1) to modulate the yield of overexpressed proteins (Smolke and Keasling 2002a); (iii) the fusion of appropriate tags to the N- or C-terminus of the overexpressed protein to facilitate

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Table 1 Copy number (molecules/cell) of plasmids commonly used to construct expression vectors Origin of replication

Plasmid prototype

Copy number

Reference

oriV

F

1–2

Jacob et al. (1963)

pSC101

pSC101

5, 6.7 ± 0.2

Hasunuma and Sekiguchi (1977), Peterson and Phillips (2008)

p15A

pACYC184

14–18

Chang and Cohen (1978), Hiszczyn´skaSawicka and Kur (1997)

ColE1

pBR322

40 ± 0.6

Lee et al. (2006)

pUC

pUC18, pUC19

411 ± 6.1

Lee et al. (2006)

its purification, increase its solubility, and eventually target its location to the periplasm (Terpe 2003); (iv) the engineering of strains and plasmids to obtain homogeneous levels of protein overexpression among the individual E. coli populations (Khlebnikov et al. 2001; Morgan-Kiss et al. 2002). In addition, overexpression systems were developed by the construction of E. coli strains (BL21(DE3) and BL21AI) able to express the RNA polymerase of phage T7 (Studier and Moffatt 1986), and by the parallel construction of expression vectors (the pET series) containing a promoter specifically recognized by T7 RNA polymerase. Remarkably, thanks to the specificity of promoter recognition by T7 RNA polymerase, high protein yields were frequently reported for E. coli BL21(DE3) hosting a pET vector. Moreover, derivatives of E. coli BL21(DE3) were isolated and successfully used to produce toxic proteins (Miroux and Walker 1996; Dumon-Seignovert et al. 2004). Accordingly, this strain and the pET vectors currently represent the standard choice for the industrial production of recombinant proteins. Nevertheless, the biology of E. coli strongly limits the type of proteins which can be overexpressed in this Gramnegative prokaryote. In particular, post-translationally modified proteins are currently produced with eukaryotic hosts, and the secretion of overexpressed proteins is poorly accomplished by E. coli compared with Gram-positive prokaryotes. This review presents recent advances in protein co-expression in E. coli, and describes how these advances could increase the

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protein types susceptible to overexpression into this industrially appealing host.

Overexpression of protein complexes The co-expression of genes was unveiled by molecular inspection of the lac operon. The lacZ, lacY, and lacA cistrons were recognized as a single transcriptional unit regulated by cis- and trans-acting molecular elements. Not surprisingly, early attempts to coexpress heterologous genes in E. coli used plasmids containing artificial operons, designed for the in vivo production of binary protein complexes. In particular, to study the assembly of cyanobacterial ribulose-1,5bisphosphate carboxylase/oxygenase, the large and small subunits of this enzyme were co-expressed in tandem (Tabita and Small 1985). This strategy was important to demonstrate that both subunits are essential for enzyme activity, and was further used to test the assembly between subunits from different species (van der Vies et al. 1986). Shortly afterwards, the biochemical nature of HIV-1 reverse transcriptase (RV) was investigated using the pKK-233-2 plasmid (Amann and Brosius 1985) to co-express a full-length and a truncated form of RV (66 and 51 kDa, respectively). Protein extracts, isolated from induced cultures and subjected to SDS-PAGE, were shown to contain both forms of RV (66 and 51 kDa), and gel filtration tests demonstrated that these extracts contained a highly active RV heterodimer (Mu¨ller et al. 1989) whose the structure was subsequently solved (Kohlstaedt et al. 1992). More recently, the availability of efficient expression tools was important to challenge the co-expression of multiple genes to yield trimeric or higher-order protein complexes, i.e. the catalytic core (Kim and McHenry 1996) and the s complex (Pritchard et al. 1996) of E. coli DNA polymerase III (Pol-III). In particular, the elegant construction of an artificial operon allowed successful overexpression of the Pol-III catalytic core composed of the a, e, and h subunits (Kim and McHenry 1996). Similarly successful was the overexpression of the s complex composed of five different subunits (s, d, d0 , v, and w) (Pritchard et al. 1996). To maximize the advantages offered by co-expression, it is desirable to express the different components of a protein complex in equimolar concentrations. To tackle this problem, Smolke and Keasling (2002a,b)

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constructed artificial dual operons containing in tandem the lacZ and gfp genes, whose transcriptional and translational products were quantified. In particular, these studies analyzed how mRNA and protein yields were affected by: (i) gene location (i.e. first or second cistron of the operon); (ii) mRNA-protecting elements (hairpins); (iii) the presence of mRNA motifs decoupling transcription and translation; (iv) gene dosage (plasmid copy number); (v) inducer concentration. Interactions among these factors were also considered, and it was shown that their appropriate manipulation can efficiently alter mRNA and protein levels (by a factor of 100 and 750, respectively). Nevertheless, it was also recognized that regulation of mRNA and protein levels is linked to intrinsic properties of the particular coding sequences subjected to overexpression (Smolke and Keasling 2002a). Therefore, when higher-order protein complexes have to be produced in vivo, it may be difficult to design artificial operons featuring good performance. Additional complications arise when attempting to co-express several genes either by traditional or tandem plasmids (Alexandrov et al. 2004; Scheich et al. 2007): (i) the molecular mass of the expression vector increases; (ii) polarity can limit the expression of genes inserted in distal positions from a single promoter. This drawback can be bypassed using independent expression plasmids to clone different genes, and cotransforming E. coli with the corresponding vectors. Over the years, a number of co-expression systems relying on couples of plasmids have been reported. Curiously, one of the first systems of this type aimed at co-expression of the two forms (66 and 51 kDa) of RV using two compatible plasmids containing the ColE1 and the p15A origin of replication, respectively (Jonckheere et al. 1996). A two-vector system was also used to co-express human CYP2D6 and NADPH-cytochrome P450 oxidoreductase (Pritchard et al. 1998). In this case, it was shown that these two proteins were expressed at similar levels (150–200 and 100–230 pmol/mg protein, respectively), and readily assembled into an active NADPHdependent monooxygenase. The promising observations reported for early coexpression experiments prompted the construction of more sophisticated plasmid sets. In particular, coexpression vectors were constructed bearing tags facilitating protein purification (Kholod and Mustelin 2001), or containing the same polylinker to allow facile gene shuttling (Dzivenu et al. 2004). Ternary co-

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expression systems were also proposed, constructing a set of three compatible plasmids containing the ColE1, p15A, or the pSC101 origin of replication (Chanda et al. 2006). The repertoire of replication origins compatible with the ColE1-type was further enriched and used for co-expression experiments in E. coli (Zhang et al. 2007; Walia et al. 2007; Zeng et al. 2010). Recently, a co-expression system whose use relies on two plasmids sharing the same polylinker and bearing the lac and araBAD regulatory elements was also reported by Conte et al. (2011). Accordingly, coexpression is triggered by the addition of IPTG and arabinose to the culture medium, and a single inducer (IPTG or arabinose) can be used to test protein production from each plasmid. More importantly, the addition of one inducer can be delayed with respect to the other, allowing facile modulation of co-expression. One of the most impressive observations reported for protein co-expression is the production of recombinant soluble hydrogenase I (SHI) from Pyrococcus furiosus in E. coli using four compatible vectors (containing the ColE1, CloDF13, RSF, or p15A origin of replication) (Sun et al. 2010). The isolated and purified recombinant SHI was shown to be active in the presence of NADPH, and featured Ni:Fe content similar to that of the native enzyme (Sun et al. 2010). The successful production of recombinant P. furiosus SHI demonstrates that protein co-expression in E. coli represents a versatile and powerful tool, whose limits might well stretch beyond our imagination.

Co-expression of post-translational modification factors In eukaryotic cells, proteome complexity is enhanced by post-translational modifications. Traditionally, it was thought that recombinant proteins bearing posttranslational modifications had to be produced using eukaryotic expression systems. E. coli indeed lacks much of the post-translational modification machinery necessary to obtain recombinant, and appropriately modified, eukaryotic proteins. Nevertheless, it was recently shown that Campylobacter jejuni, a Gramnegative bacterium, produces N-glycosylated proteins (Szymanski et al. 1999; Linton et al. 2002). Shortly afterwards, it was demonstrated that co-expression is an efficient tool to enrol in E. coli the glycosylation system of C. jejuni (Wacker et al. 2002). This was

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done by cloning the whole protein glycosylation (pgl) locus from C. jejuni into pACYC184, and co-expressing the target AcrA protein (a transmembrane transporter) from a compatible plasmid, pET24b (Wacker et al. 2002). Among the genes of the pgl cluster, pglB was later shown to be the essential determinant of protein glycosylation (Feldman et al. 2005). Accordingly, two compatible vectors were used to express the target protein and pglB, and a third compatible plasmid was used to co-express the enzymes necessary to yield in E. coli heterologous O antigens (the glycan polymers linked to the lipopolysaccharide, LPS, of the outer membrane of Gram-negatives), such as that from Shigella dysenteriae (Ihssen et al. 2010). This strategy was used to produce glycoconjugated vaccines in E. coli (Ihssen et al. 2010). Alternatively, the glycosylation tag DQNAT can be fused to target proteins to obtain their glycosylation (Fisher et al. 2011). Furthermore, co-expression was used to produce in E. coli glycosylated scFv antibody fragments with better solubility and stability than the unmodified antibodies (Lizak et al. 2011). Recently, Campylobacter lari was also shown to contain functional protein glycosylation machinery (Schwarz et al. 2010). Interestingly, the two glycosylation systems from C. jejuni and C. lari feature different specificities (Schwarz et al. 2010), and these different posttranslational modification preferences were shown to persist in E. coli (Schwarz et al. 2010). This and previous findings indicate that the production of glycosylated recombinant proteins in E. coli, whose feasibility relies on protein co-expression, now seems promising. Co-expression was also used to produce eukaryotic phosphorylated proteins in E. coli. By co-expressing a eukaryotic protein (mouse or human Jun dimerization protein 2) and a kinase (e.g. human Jun N-terminal kinase 1, JNK1), it was demonstrated that phosphorylation of the target occurs in E. coli (Murata et al. 2008). Also human proteins, produced and phosphorylated in E. coli, feature phosphorylation-dependent interaction capabilities (Murata et al. 2008). Interestingly, the specific phosphorylation of a protein domain in E. coli was also reported (Sugase et al. 2008). Protein methylation occurs at the expense of a target lysine (K), and several lysine methyltransferases have been identified. By co-expressing calmodulin methyltransferase and members of a library of calmodulin variants, Magnani et al. (2012) showed

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that trimethylation of calmodulin K115 occurs in E. coli. Moreover, trimethylation affected both the thermostability and allosteric response to calcium of this protein (Magnani et al. 2012). Proteins are myristoylated via the ligation of myristic acid to the N-terminal glycine of the target. This reaction is catalyzed by N-myristoyltransferases (NMTs) whose co-expression in E. coli yields recombinant myristoylated proteins (Duronio et al. 1990; Ray et al. 1992; Ames et al. 1994; Ladant 1995; Breuer et al. 2006; Lim et al. 2009; Liu et al. 2009). To this aim, two compatible plasmids are generally used and contain the gene coding for the NMT and the target protein, respectively. Recently, myristoylated Nef protein was produced from HIV-1, and purified Nef did exclusively contain the myristoylated protein (Glu¨ck et al. 2010). The majority of eukaryotic proteins bear an acetylated N-terminus (Arnesen et al. 2009). This posttranslational modification is important for the stability and proper function of several eukaryotic targets. Interestingly, Fang et al. (2009) showed that the E. coli gene rimJ codes for an N-ter acetylase (Nat). Moreover, co-expression of this enzyme and a target peptide (recombinant a1-thymosin) is an effective approach for the production of acetylated proteins in E. coli (Ren et al. 2011). Acetylated proteins can also be produced in E. coli by co-expressing a Nat complex from Schizosaccharomyces pombe and target proteins, i.e. murine and S. pombe a-tropomyosin, human Spartin, and Saccharomyces cerevisiae Tfs1 (Johnson et al. 2010). This co-expression system produced some remarkable observations: (i) the target proteins were acetylated in E. coli, with modification yields ranging from 25 (Spartin) to 100 % (S. pombe a-tropomyosin); (ii) acetylation in E. coli of both human and yeast atropomyosin increased the functionality of these proteins, i.e. their actin-binding competence; (iii) the recombinant protein yield was higher under acetylating conditions, suggesting that this post-translational modification enhances the stability of overexpressed proteins (Johnson et al. 2010). The ligation of ubiquitin or small ubiquitin-related modifier (SUMO, 11 kDa) to eukaryotic proteins regulates the activity and stability of several targets. Remarkably, co-expression was reported to afford the sumoylation of recombinant proteins in E. coli (O’Brien and DeLisa 2012). To this aim, it was necessary to use three compatible plasmids featuring

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different copy numbers, i.e. bearing the ColE1, p15A or pSCS101 origin of replication (Table 1) to overexpress the target protein and SUMO, SUMO E3 ligase, SUMO E1 and SUMO E2 ligases, respectively (O’Brien and DeLisa 2012). The yields of sumoylated proteins were higher when all three SUMO ligases were co-expressed, in comparison with yields observed in the presence of SUMO ligases E1 and E2 alone. The co-expression of genes in E. coli is not only an appropriate tool for the production of proteins bearing ‘‘canonical’’ post-translational modifications, but can also be used to produce proteins bearing ‘‘functional’’ post-translational modifications. To obtain a fully functional recombinant protein, Choi et al. (2012) coexpressed in E. coli a mussel adhesive protein (MAP) and Streptomyces antibioticus tyrosinase. MAPs are rich in tyrosines, some of which are converted by hydroxylation into 3,4-dihydroxy-phenylalanine (DOPA). The presence of DOPA in MAPS is known to confer the adhesive properties to these proteins. To produce DOPA-containing MAPs, Choi et al. coexpressed a MAP chimaera (MAP1-MAP5-MAP1) and S. antibioticus tyrosinase, along with a protein factor (ORF 438) essential for the incorporation of copper into tyrosinase (Choi et al. 2012). Using this strategy, they demonstrated that the MAP chimaera did contain dopaquinone (the oxidation product of DOPA) in substitution of a specific tyrosine, and featured higher solubility and adhesive strength than the unmodified protein.

Co-expression and expansion of the 20-letter alphabet Natural proteins are coded by a 20-letter alphabet, possibly enriched by selenocysteine and selenomethionine. Notably, the artificial synthesis of peptides can take advantage of unnatural amino acids, conferring particular chemical properties to their hosts. Moreover, the use of D-amino acids sustains the construction of unnatural secondary structure elements, such as the right-handed alpha helix (Shepherd et al. 2009). Nevertheless, the difficulties of chemical synthesis multiply dramatically with the increase in molecular mass of the desired protein. Accordingly, the biotechnological use of an expanded alphabet of amino acids for the production of recombinant proteins would

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significantly enrich protein complexity and function. Co-expression of proteins and tRNAs provides an elegant solution to this challenge. Pioneering efforts by Schultz and coworkers demonstrated that an artificial suppressor tRNA (sup-tRNA) can be chemically aminoacylated and successfully used to incorporate unnatural amino acids during protein synthesis in vitro (Noren et al. 1989). These important observations prompted the search for the components necessary for the in vivo production of proteins bearing unnatural amino acids. The search for these components focused on heterologous aminoacyl tRNA synthetases (ATSs) and tRNAs which could be safely expressed in E. coli without impairing host physiology. This is relevant for the production of recombinant proteins bearing unnatural amino acids considering that: (i) to incorporate unnatural amino acids into recombinant proteins in vivo, one of the three stop codons (UAG, UGA, UAA) can be converted to a coding signal; (ii) to convert a stop codon to a coding signal an ATS/tRNA special pair has to be constructed and expressed; (iii) the natural stop codons of the bacterial host must not be suppressed by the special ATS/tRNA pair; (iv) neither the ATS nor the tRNA of the special pair must interact with the decoding elements of the host (the heterologous ATS should not interact with natural tRNAs, and the heterologous tRNA should not be aminoacylated by native ATSs). To avoid suppression by special ATS/tRNA pairs, Wang et al. (2001) co-expressed into E. coli a heterologous and appropriately mutated couple from Methanococcus jannaschii, and successfully used this system to incorporate O-methyl-L-tyrosine into E. coli dihydrofolate reductase. Subsequently, a number of systems were reported for the expression in E. coli of recombinant proteins bearing unnatural amino acids (for a review see Young and Schultz 2010). These systems include the construction of a special ATS/ tRNA couple derived from Pyrococcus horikoshii and able to decode nucleotide quadruplets, i.e. the artificial codon AGGA (Anderson and Schultz 2003; Anderson et al. 2004). This system afforded the incorporation of L-homoglutamine into sperm whale myoglobin. Moreover, when the M. jannaschii and P. horikoshii special pairs were co-expressed (Anderson et al. 2004), both O-methyl-L-tyrosine and L-homoglutamine were incorporated into myoglobin, coded by a gene containing the AGGA quadruplet at position 24, and the

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UAG suppressible amber codon at position 75 (Fig. 1). Under these conditions, the yield of myoglobin containing both unnatural amino acids was equal to 1.7 mg/l (Anderson et al. 2004). However, the performances of ATS/tRNA special pairs are likely susceptible to significant improvements, and new coexpression tools could further enhance the yield of proteins bearing unnatural amino acids and produced in vivo in E. coli.

Co-expression and protein secretion Seven distinct protein secretion systems have been identified in prokaryotes (for recent reviews, see Abdallah et al. 2007; Douzi et al. 2012; Jong et al. 2010; Korotkov et al. 2012; Marlovits and Stebbins 2010; Silverman et al. 2012; Zechner et al. 2012). It

Fig. 1 Co-expression of genes in E. coli and production of recombinant proteins containing unnatural amino acids. The gene coding for myoglobin from sperm whale, to which codons 24 and 75 were substituted with AGGA and UAG, respectively, was expressed from a p15A-type plasmid (Anderson et al. 2004). Two special suppressing tRNAs (tRNAUCCU and tRNACUA) were also expressed from the same plasmid. A

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has also been established that some of these secretion machineries are relatively simple systems (e.g. the type I and type V systems), while the functionality of others relies on a number of components. Taking advantage of the simplicity of the type I secretion system, secreted heterologous proteins in E. coli were produced as early as 1992 (Suh and Benedik 1992). In this case, it was recognized that the C-terminus of Serratia marcescens metalloprotease bears a LXGGXGND motif, the determinant for secretion of a-hemolysin (HlyA). Co-expression of the HlyB and HlyD secretion factors provided the means to overexpress the metalloprotease in the extracellular medium (Fig. 2). Later work by Gentschev et al. demonstrated that proteins naturally devoid of the LXGGXGND motif can be overexpressed and secreted by E. coli (Gentschev et al. 1996). To this aim, expression vectors were constructed to obtain the

compatible pUC derivative was used to co-express the cognate homoGln-tRNAUCCU and O-methyl-Tyr-tRNACUA synthetases from P. horikoshii and M. jannaschii, respectively. The production of full-length myoglobin was fully dependent on the presence of L-homoglutamine and O-methyl-L-tyrosine in the culture medium (Anderson et al. 2004)

Biotechnol Lett (2013) 35:1971–1981

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fusion of the C-ter of a target heterologous protein to the 61 C-ter amino acids of a-hemolysin. The proteins of interest, the HlyB and HlyD secretion factors, were co-expressed in tandem using a single vector (pX1), or co-expressed from a compatible plasmid (pBD152). In this case, secretion yields ranging from 20 to 60 % were reported (Gentschev et al. 1996). However, the C-terminus of the overexpressed and secreted proteins did retain the fused 61 residues of a-hemolysin. A similar approach was used to overexpress and secrete scFv in E. coli (Fernandez et al. 2000). More recently,

the C-ter motif of Pseudomonas sp. lipase (GGXGXD XUX) was used to produce fusions with proteins to be overexpressed and secreted in E. coli (Angkwidjaja et al. 2006). Co-expression of the lipBCD genes from S. marcescens provided the means to produce and efficiently secrete alkaline phosphatase into the culture medium (Angkwidjaja et al. 2006). This strategy was further investigated (Chung et al. 2009) by fusing the C-ter of target proteins to the secretion/chaperon domain of Pseudomonas fluorescens thermostable lipase (TliA). To overexpress these

Fig. 2 Co-expression of genes and secretion of recombinant proteins in E. coli. The type I secretion system was used in E. coli to produce a metalloprotease from S. marcescens (Suh and Benedik 1992). This protease was overexpressed from of a ColE1-type vector, and the HlyB and HlyB hemolysin transporters were co-expressed by means of a compatible p15A-type plasmid. The endogenous TolC transporter afforded secretion of the metalloprotease into the extracellular medium

(Suh and Benedik 1992). The secretion of chitinase, overexpressed in E. coli from a pUC derivative, was obtained coexpressing (using a compatible p15A-type vector) the gsp locus, coding for a type II secretion system (Francetic et al. 2000). The overexpressed chitinase, bearing a signal sequence (blue sphere) is transported across the inner membrane via the Sec translocon. Subsequently, the signal peptide is cleaved and the chitinase secreted through the GspCD transporter

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target proteins in the extracellular medium, the PrtDEF or the TliDEF transporter (from Erwinia chrysanthemi and from P. fuorescens, respectively) were co-expressed (Chung et al. 2009). Secretion was observed for fusion proteins containing the factor Xa protease consensus sequence, located between the TliA secretion/chaperon domain and the target protein (Chung et al. 2009). Therefore, the TliA domain could eventually be removed from the target protein using the factor Xa protease. Although featuring a rather complicated architecture, the type II secretion system was successfully used to overexpress and secrete chitinase in E. coli (Francetic et al. 2000). In particular, the endogenous gsp locus coding for the components of this secretion system was cloned into a p15A expression vector and the chiA gene, coding for chitinase, was cloned into a pUC derivative (Fig. 2). Interestingly, heterologous genes coding for the type II secretion system were also used to secrete overexpressed proteins in E. coli. The out genes of Erwinia chrysanthemi were indeed cloned into a cosmid, pCPP2006, and the pelE gene, coding for E. chrysanthemi pectate lyase, was cloned into a ColE1type vector (He et al. 1991). Under these conditions, 90 % of pectate lyase was detected in the extracellular medium. Later work demonstrated that endoglucanase from E. chrysanthemi can also be overexpressed and secreted from E. coli (Zhou et al. 1999). In this case, using pCPP2006 and a pUC derivative hosting the endoglucanase-coding gene celZ, up to 74 % of the enzyme was secreted into the medium. Independently of co-expression, type III (Majander et al. 2005; Blanco-Toribio et al. 2010) and type V (Sevastsyanovich et al. 2012) secretion systems were used to overexpress and secrete heterologous proteins in E. coli. In addition, type V secretion systems were used to display heterologous proteins on the cellular surface of E. coli (for a review see Jong et al. 2010).

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of membrane proteins overexpressed in E. coli. Nannenga and Baneyx (2011b) analyzed the effect of coexpressing different molecular chaperones on the production level of heterologous membrane proteins in E. coli. As a first test, they constructed strains bearing a deletion of tig, the gene coding for the socalled trigger factor molecular chaperone. Surprisingly, the deletion of tig significantly increased the overexpression level of the endogenous histidine kinase Zras, and two heterologous rhodopsins, i.e. Haloterrigena turkmenica deltarhodpsin (Htdr) and Natronobacterium pharaonis sensory rhodopsin II (NpsrII). To further increase the expression level of these membrane proteins, Nannenga and Baneyx (2011a) tested the co-expression of two molecular chaperones, i.e. signal recognition particle (SRP) and YidC, both in Tig? and in Dtig genetic backgrounds. To this aim, they used a pACYC184 derivative (pMM102) to express the molecular chaperones, and a compatible plasmid (ColE1-type) to co-express Zras, Htdr, or NpsrII. The yield of each membrane protein was quantitatively estimated under different conditions, producing the following observations: (i) deletion of tig increased the expression level of Zras, Htdr, and NpsrII; (ii) co-expression of SRP decreased the yield of the three membrane proteins considered; (iii) co-expression of YidC increased the expression level of NpsrII, and this effect was additive with the increase due to the deletion of tig (Nannenga and Baneyx 2011a). In addition: (i) tig deletion and YidC co-expression increased the yield of NpsrII up to one order of magnitude; (ii) YidC was co-expressed under the regulation of its natural promoter. Accordingly, the co-expression of YidC might be tuned (e.g. using strains featuring homogeneous induction) to further increase the yield of membrane proteins overexpressed in E. coli.

Conclusions Co-expression and overexpression of membrane proteins The overexpression of integral membrane proteins is a difficult task (Drew et al. 2006). In particular, the misfolding and/or aggregation of membrane proteins have to be prevented to achieve significant production levels. Interestingly, recent work indicates that coexpression is an appropriate tool to increase the yield

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The development of co-expression systems is significantly expanding the landscape of recombinant protein production afforded by E. coli. In particular, the use of co-expression to obtain protein complexes assembled in vivo and containing a multitude of subunits is a wellestablished technique. In addition, it was convincingly demonstrated that co-expression can be used in E. coli to produce proteins bearing a variety of

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post-translational modifications or unnatural amino acids. Further, co-expression is recently gaining attention to construct E. coli strains competent in protein secretion, and to achieve high production levels of heterologous membrane proteins. For these important challenges further improvements will likely be reported in the near future, reshaping the use of E. coli as a platform for the production of recombinant proteins. Acknowledgments We are indebted to Anne Prudence Collins (Koine` SaS, Editorial Service for Academic Publications) for language editing.

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