Bacterial-based membrane protein production

Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression

The formation of inclusion bodies (IBs) in recombinant protein biotechnology has become one of the most frequent undesirable occurrences in both research and industrial applications. So far, the pET System is the most powerful system developed for the production of recombinant proteins when Escherichia coli is used as the microbial cell factory. Also, using fusion tags to facilitate detection and purification of the target protein is a commonly used tactic. However, there is still a large fraction of proteins that cannot be produced in E. coli in a soluble (and hence functional) form. Intensive research efforts have tried to address this issue, and numerous parameters have been modulated to avoid the formation of inclusion bodies. However, hardly anyone has noticed that adding fusion tags to the recombinant protein to facilitate purification is a key factor that affects the formation of inclusion bodies. To test this idea, the industrial biocatalysts uridine phosphorylase from Aeropyrum pernix K1 and (+)-γ-lactamase and (?)-γ-lactamase from Bradyrhizobium japonicum USDA 6 were expressed in E. coli by using the pET System and then examined. We found that using a histidine tag as a fusion partner for protein expression did affect the formation of inclusion bodies in these examples, suggesting that removing the fusion tag can promote the solubility of heterologous proteins. The production of soluble and highly active uridine phosphorylase, (+)-γ-lactamase, and (?)-γ-lactamase in our results shows that the traditional process needs to be reconsidered. Accordingly, a simple and efficient structure-based strategy for the production of valuable soluble recombinant proteins in E. coli is proposed.     

Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography

Immobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and YfbG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.     

SuptoxD2.0: A second-generation engineered Escherichia coli strain achieving further enhanced levels of recombinant membrane protein production

The bacterium Escherichia coli is among the most popular hosts for recombinant protein production, including that of membrane proteins (MPs). We have recently generated the specialized MP-producing E. coli strain SuptoxD, which upon co-expression of the effector gene djlA, is capable of alleviating two major bottlenecks in bacterial recombinant MP production: it suppresses the toxicity that frequently accompanies the MP-overexpression process and it markedly increases the cellular accumulation of membrane incorporated and properly folded recombinant MP. Combined, these two positive effects result in dramatically enhanced volumetric yields for various recombinant MPs of both prokaryotic and eukaryotic origin. Based on the observation that djlA is found in the genomes of various pathogenic bacteria, the aim of the present work was to investigate (a) whether other naturally occurring DjlA variants can exert the MP toxicity-suppressing and production-promoting effects similarly to the E. coli DjlA and (b) if we can identify a DjlA variant whose efficiency surpasses that of the E. coli DjlA of SuptoxD. We report that a quite surprisingly broad variety of homologous DjlA proteins exert beneficial effects on recombinant MP when overexpressed in E. coli. Furthermore, we demonstrate that the Salmonella enterica DjlA is an even more potent enhancer of MP productivity compared with the E. coli DjlA of SuptoxD. Based on this, we constructed a second-generation SuptoxD strain, termed SuptoxD2.0, whose MP-production capabilities surpass significantly those of the original SuptoxD, and we anticipate that SuptoxD2.0 will become a broadly utilized expression host for recombinant MP production in bacteria.     

A unified method for purification of basic proteins

Protein purification is still very empirical, and a unified method for purifying proteins without an affinity tag is not available yet. In the postgenomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied for purifying any recombinant basic protein from Escherichia coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI−1 ? 6.94) may be purified from E. coli in a single step using a cation-exchanger resin, SP-Sepharose, and a selected buffer pH, depending on the pI of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid-interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single step.     

A Pair of Ligation-Independent Escherichia coli Expression Vectors for Rapid Addition of a Polyhistidine Affinity Tag to the N- or C-Termini of Recombinant Proteins

6×His tag is one of the most widely used affinity fusion tags that facilitates detection and purification of recombinant proteins. However, the location of this tag within a particular type of protein may influence the expression, solubility, and bioactivity of the protein, and the optimal location needs to be determined experimentally. To provide a tool for rapid generation of 6× His tags at the N- or C-terminus of any recombinant protein, we have constructed a pair of Escherichia coli expression vectors—pLIC-NHis and pLIC-CHis—based on the pET30a vector, for ligation-independent cloning (LIC). Construction of this new pair of LIC vectors was accomplished by replacement of the multiple cloning site of pET30a with two specifically designed LIC cloning sites. A target gene derived by PCR with a pair of predesigned primers can be inserted into the LIC site of pLIC-NHis for expression of recombinant proteins fused with the N-terminal sequence MHHHHHHG or into that of pLIC-CHis for expression of recombinant proteins with the C-terminal sequence THHHHHH. Successful expression of two normal mammalian prion proteins and five bacterial proteins in E. coli using this pair of LIC vectors reveals that these vectors are valuable tools for the production of recombinant His-tagged proteins in E. coli.     

Conversion of a Chaperonin GroEL-independent Protein into an Obligate Substrate

Chaperones assist protein folding by preventing unproductive protein aggregation in the cell. In Escherichia coli, chaperonin GroEL/GroES (GroE) is the only indispensable chaperone and is absolutely required for the de novo folding of at least ∼60 proteins. We previously found that several orthologs of the obligate GroE substrates in Ureaplasma urealyticum, which lacks the groE gene in the genome, are E. coli GroE-independent folders, despite their significant sequence identities. Here, we investigated the key features that define the GroE dependence. Chimera or random mutagenesis analyses revealed that independent multiple point mutations, and even single mutations, were sufficient to confer GroE dependence on the Ureaplasma MetK. Strikingly, the GroE dependence was well correlated with the propensity to form protein aggregates during folding. The results reveal the delicate balance between GroE dependence and independence. The function of GroE to buffering the aggregation-prone mutations plays a role in maintaining higher genetic diversity of proteins.     

Does Lack of Secondary Structure Imply Intrinsic Disorder in Proteins? A Sequence Analysis

Intrinsically disordered proteins (IDPs)/protein regions (IDPRs) lack unique three-dimensional structure at the level of secondary and/or tertiary structure and are represented as an ensemble of interchanging conformations. To investigate the role of presence/absence of secondary structures in promoting intrinsic disorder in proteins, a comparative sequence analysis of IDPs, IDPRs and proteins with minimal secondary structures (less than 5%) is required. A sequence analysis reveals proteins with minimal secondary structure content have high mean net positive charge, low mean net hydrophobicity and low sequence complexity. Interestingly, analysis of the relative local electrostatic interactions reveal that an increase in the relative repulsive interactions between amino acids separated by three or four residues lead to either loss of secondary structure or intrinsic disorder. IDPRs show increase in both local negative-negative and positive-positive repulsive interactions. While IDPs show a marked increase in the local negative-negative interactions, proteins with minimal secondary structure depict an increase in the local positive-positive interactions. IDPs and IDPRs are enriched in D, E and Q residues, while proteins with minimal secondary structure are depleted of these residues. Proteins with minimal secondary structures have higher content of G and C, while IDPs and IDPRs are depleted of these residues. These results confirm that proteins with minimal secondary structure have a distinctly different propensity for charge, hydrophobicity, specific amino acids and local electrostatic interactions as compared to IDPs/IDPRs. Thus we conclude that lack of secondary structure may be a necessary but not a sufficient condition for intrinsic disorder in proteins.     

Characterisation of the interaction of colicin A with its co-receptor TolA

Colicin A enters Escherichia coli cells through interaction with endogenous TolA and TolB proteins. In vitro, binding of the colicin A translocation domain to TolA leads to unfolding of TolA. Through NMR studies of the colicin A translocation domain and polypeptides representing the individual TolA and TolB binding epitopes of colicin A we question if the unfolding of TolA induced by colicin A is likely to be physiologically relevant. The NMR data further reveals that the colicin A binding site on TolA is different from that for colicin N which explains why there is a difference in colicin toxicity for E. coli carrying a TolA-III homologue from Yersina enterocolitica in place of its own TolA-III.

Structured summary

MINT-7888512: TolA (uniprotkb:P19934) and Col-A (uniprotkb:P04480) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7888526: TolA (uniprotkb:P19934) and TolB (uniprotkb:P0A857) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7888999: TolA (uniprotkb:P19934), TolB (uniprotkb:P0A855) and Col-A (uniprotkb:P04480) physically interact (MI:0915) by molecular sieving (MI:0071)MINT-7888982: TolA (uniprotkb:P19934), TolB (uniprotkb:P0A855) and Col-A (uniprotkb:P04480) physically interact (MI:0915) by nuclear magnetic resonance (MI:0077)     

Strategies for the Production of Recombinant Protein in Escherichia coli

In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins.     

Export of a hyperexpressed mammalian globular cytochrome b5 precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond

A chimeric mammalian globular cytochrome b₅ fused to Escherichia coli alkaline phosphatase signal sequence (SS) was used as a model probe to investigate the influence of substituting each one of the standard 20 amino acids at its N‐terminus on the Sec‐dependent export of the precursor to the periplasmic space of E. coli. Substituting the native Met⁺¹ of the passenger protein flanking the SS with any one of the remaining 19 amino acids introduced significant changes in the export of cytochrome b₅ without jamming the Sec‐dependent translocon. Acidic and hydrophilic residues proved to be the most efficient promoters of export. Small, nonbulky and basic residues yielded intermediate levels of the hemoprotein export. Replacement with a Cys⁺¹ residue generated significant quantities of both monomeric and disulfide‐linked dimeric forms. However, bulky, aromatic and hydrophobic residues caused a significant decline in the rates of secretion. In expectation with their absences in the natural periplasmically secreted proteins, Pro and Ile‐tagged cytochrome b₅ precursors failed to generate any detectable secreted recombinant products. Although Ala, amongst the native E. coli periplasmic proteins, is the preferred X⁺¹ residue with an occurrence of 50% frequency, it proved half as effective in promoting export when inserted proximally to the SS of cytochrome b₅. The mechanisms involved for these export variations are discussed. The findings will prove beneficial for high‐level generation of recombinant proteins by secretory means for pharmaceutical and related biotechnological applications.     

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)₆-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni²⁺-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.     

Expanding the landscape of recombinant protein production in Escherichia coli

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 co-expression 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 co-expression to overproduce membrane proteins.     

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

An auto-inducible expression system based on the RhlI-RhlR quorum-sensing regulon for recombinant protein production in <Emphasis Type="Italic">E. coli</Emphasis>

An artificial network which can accomplish recombinant protein synthesis guided by cell population in E. coli was constructed. The successful functioning of this network requires two plasmids, pWNB and pET. pWNB is responsible for production of T7 RNA polymerase, which controls pET; pET, in turn, regulates the production of target proteins. Several model proteins were tested and the results show that this E. coli system can be used to efficiently express various recombinant proteins. Since system contains T7 RNA polymerase production elements, it is transferable and applicable to well-characterized E. coli strains. Compared to the IPTG-induced system, an equal or greater amount of target protein can be obtained using this auto-inducible expression system in flasks and bioreactors. Our results suggest that it is a competitive alternative to other expression systems used in labs or for industrial applications.     

Practical protocols for production of very high yields of recombinant proteins using Escherichia coli

The gram‐negative bacterium Escherichia coli offers a mean for rapid, high yield, and economical production of recombinant proteins. However, high‐level production of functional eukaryotic proteins in E. coli may not be a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in E. coli have been explored for host strain selection, plasmid copy numbers, promoter selection, mRNA stability, and codon usage, significantly enhancing the yields of the foreign eukaryotic proteins. We have been working on optimizations of bacterial expression conditions and media with a focus on achieving very high cell density for high‐level production of eukaryotic proteins. Two high‐cell‐density bacterial expression methods have been explored, including an autoinduction introduced by Studier (Protein Expr Purif 2005;41:207–234) recently and a high‐cell‐density IPTG‐induction method described in this study, to achieve a cell‐density OD₆₀₀ of 10–20 in the normal laboratory setting using a regular incubator shaker. Several practical protocols have been implemented with these high‐cell‐density expression methods to ensure a very high yield of recombinant protein production. With our methods and protocols, we routinely obtain 14–25 mg of NMR triple‐labeled proteins and 17–34 mg of unlabeled proteins from a 50‐mL cell culture for all seven proteins we tested. Such a high protein yield used the same DNA constructs, bacterial strains, and a regular incubator shaker and no fermentor is necessary. More importantly, these methods allow us to consistently obtain such a high yield of recombinant proteins using E. coli expression.     

Obtaining highly purified intrinsically disordered protein by boiling lysis and single step ion exchange

Intrinsically disordered proteins (IDPs) is a term used to describe proteins that do not have a well-defined tertiary structure. IDPs have many roles such as in cell cycle control (p53), neuronal signal transmission (myelin basic protein), and protein stability (dehydrins). Producing recombinant IDPs in bacteria for nuclear magnetic resonance (NMR) studies is problematic because the lack of stable tertiary structure makes them excellent substrates for bacterial proteases, which will cause loss in yield. We have developed a two-step method to produce the grape dehydrin K₂ and YSK₂ using Escherichia coli. Dehydrins are expressed by certain plants in response to dehydration, increased salinity, or low temperatures. Purification of 10 mg/L (K₂) and 15 mg/L (YSK₂) was performed by boiling bacterial pellets to lyse the cells, remove most of the contaminating proteins, and denature bacterial proteases. This resulted in protein purity comparable to that produced by sonication and nickel affinity chromatography. Boiling was followed by cation exchange chromatography to remove the remaining trace contaminants. The sample was shown to be more than 95% pure by reversed-phase high-performance liquid chromatography. The method presented here can easily be adapted to the purification of other IDPs and heat-stable proteins without requiring multiple chromatography steps or the use of protease inhibitors.     

Efficient genetic approaches for improvement of plasmid based expression of recombinant protein in Escherichia coli: A review

During the past two decades, there has been an explosion of new knowledge and techniques in the field of recombinant protein expression. However, over-expression of “difficult to express proteins” with therapeutic importance continues to be a challenging task for successful commercialization of these proteins. With the emergence of the bio-similar market, enhancing the efficiencies of the production process has become a critical factor in the commercial viability of novel products. Despite the availability of numerous technological advancements, recombinant protein expression in Escherichia coli remains difficult. Therefore, addressing upstream bottlenecks in combination with genetically modified expression hosts could be a viable strategy to enhance production. Problems like poor expression, plasmid instability, protein aggregation, protein degradation, and metabolic stress associated with recombinant protein production need special consideration during bioprocess development at bioreactor level. However, a comprehensive universal strategy for attaining efficient expression in E. coli seems unrealistic and must be resolved empirically. In this review, we have discussed some common problems and their apparent solutions for plasmids based recombinant gene expression in E. coli.     

Preparation of cell-permeable Cre recombinase by expressed protein ligation

Background

Protein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli.

Results

Expressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC.

Conclusions

Collectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0126-z) contains supplementary material, which is available to authorized users.