Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA

The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding. However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding. In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E. coli. We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main catalyst of disulfide bond formation in E. coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation. To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives. We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium. The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.     

Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells

An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.     

Scalable purification of Bacillus anthracis protective antigen from Escherichia coli

The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli. The rPA protein was produced in a 50-L fermentor and purified to >99% purity using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatography. The final yield of purified rPA from medium cell density fermentations resulted in approximately 2.7 g of rPA per kg of cell paste (approximately 270 mg/L) of highly pure, biologically active rPA protein. The results presented here exhibit the ability to generate multi-gram quantities of rPA from E. coli that may be used for the development of new anthrax vaccines and anthrax therapeutics.     

粪产碱杆菌青霉素G酰化酶在枯草芽孢杆菌中的表达、纯化及其性质

利用PCR技术克隆了粪产碱杆菌 (Alcaligenesfaecalis,CICCAS1.76 7)青霉素G酰化酶 (pencillinGacylase ,PGA)基因 (GenBank登录号AF4 5 5 35 6 )。通过构建工程菌E .coli(pETAPGA) ,该酶在大肠杆菌中获得了表达 ,表达产物分泌到周质空间。进一步构建的工程菌B .subtilis (pMAPGA)和B .subtilis(pBAPGA)实现了该酶的胞外分泌表达。分泌表达的最高表达量为 6 5 3u/L ,比野生型A .faecalis表达量高 10 9倍。表达产物经硫酸铵分级沉淀和DEAE SepharoseCL 6B两步纯化 ,纯度提高 86倍 ,活力回收率达到 81% ,纯化后的PGA活力为 1.4 6 9u/mg。研究表明 ,PGA家族成员中只有粪产碱杆菌PGA和巨大芽孢杆菌PGA可以在枯草芽孢杆菌中分泌表达。与巨大芽孢杆菌PGA相比 ,粪产碱杆菌PGA的最适pH值为 8.0 ,最适温度为 6 0°C ,而且在有机溶剂中具有更强的稳定性。该酶在水相中具有较低的头孢氨苄合成活力。本研究为粪产碱杆菌PGA的获得提供了新的途径。     

Construction and Characterization of Versatile Cloning Vectors for Efficient Delivery of Native Foreign Proteins to the Periplasm ofEscherichia coli

Induction of the wild type cholera toxin operon (ctxAB) from multicopy clones inEscherichia coliinhibited growth and resulted in low yields of cholera toxin (CT). We found that production of wild type CT or its B subunit (CT-B) as a periplasmic protein was toxic forE. coli,but by replacing the native signal sequences of both CT-A and CT-B with the signal sequence from the B subunit ofE. coliheat-labile enterotoxin LTIIb we succeeded for the first time in producing CT holotoxin in high yield inE. coli.Based on these findings, we designed and constructed versatile cloning vectors that use the LTIIb-B signal sequence to direct recombinant native proteins with high efficiency to the periplasm ofE. coli.We confirmed the usefulness of these vectors by producing two other secreted recombinant proteins. First, usingphoAfromE. coli,we demonstrated that alkaline phosphatase activity was 17-fold greater when the LTIIb-B signal sequence was used than when the native leader for alkaline phosphatase was used. Second, using thepspAgene that encodes pneumococcal surface protein A fromStreptococcus pneumoniae,we produced a 299-residue amino-terminal fragment of PspA inE. coliin large amounts as a soluble periplasmic protein and showed that it was immunoreactive in Western blots with antibodies against native PspA. The vectors described here will be useful for further studies on structure–function relationships and vaccine development with CT and PspA, and they should be valuable as general tools for delivery of other secretion-competent recombinant proteins to the periplasm inE. coli.     

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.     

Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines.

Using the well-characterized antibody McPC603 as a model, we had found that the Fv fragment can be isolated from Escherichia coli as a functional protein in good yields, whereas the amount of the correctly folded Fab fragment of the same antibody produced under identical conditions is significantly lower. In this paper, we analyse the reasons for this difference. We found that a variety of signal sequences function in the secretion of the isolated chains of the Fab fragment or in the co-secretion of both chains in E.coli. The low yield of functional Fab fragment is not caused by inefficient expression or secretion in E.coli, but by inefficient folding and/or assembly in the periplasm. We compared the folding yields for the Fv and the Fab fragment in the periplasm under various conditions. Several diagnostic framework variants were constructed and their folding yields measured. The results show that substitutions affecting cis-proline residues and those affecting various disulphide bonds in the protein are by themselves insufficient to dramatically change the partitioning of the folding pathway to the native structure, and the cause must lie in a facile aggregation of folding intermediates common to all structural variants. However, all structural variants could be obtained in native form, demonstrating the general utility of the secretory expression strategy.     

DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo

Oxidative protein folding in the periplasm of Escherichia coli is catalyzed by the thiol-disulfide oxidoreductases DsbA and DsbC. We investigated the catalytic efficiency of these enzymes during folding of proteins with a very complex disulfide pattern in vivo and in vitro, using the Ragi bifunctional inhibitor (RBI) as model substrate. RBI is a 13.1 kDa protein with five overlapping disulfide bonds. We show that reduced RBI can be refolded quantitatively in glutathione redox buffers in vitro and spontaneously adopts the single correct conformation out of 750 possible species with five disulfide bonds. Under oxidizing redox conditions, however, RBI folding is hampered by accumulation of a large number of intermediates with non-native disulfide bonds, while a surprisingly low number of intermediates accumulates under optimal or reducing redox conditions. DsbC catalyzes folding of RBI under all redox conditions in vitro, but is particularly efficient in rearranging buried, non-native disulfide bonds formed under oxidizing conditions. In contrast, the influence of DsbA on the refolding reaction is essentially restricted to reducing redox conditions where disulfide formation is rate limiting. The effects of DsbA and DsbC on folding of RBI in E.coli are very similar to those observed in vitro. Whereas overexpression of DsbA has no effect on the amount of correctly folded RBI, co-expression of DsbC enhanced the efficiency of RBI folding in the periplasm of E.coli about 14-fold. Addition of reduced glutathione to the growth medium together with DsbC overexpression further increased the folding yield of RBI in vivo to 26-fold. This shows that DsbC is the bacterial enzyme of choice for improving the periplasmic folding yields of proteins with very complex disulfide bond patterns.     

Periplasmic production of native human proinsulin as a fusion to E. coli ecotin

Native proinsulin belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its small size, a high proteolytic decay, and the necessity to form a native disulfide pattern. In the present study, human proinsulin was produced in the periplasm of E. coli as a fusion to ecotin, which is a small periplasmic protein of 16 kDa encoded by the host, containing one disulfide bond. The fusion protein was secreted to the periplasm and native proinsulin was determined by ELISA. Cultivation parameters were studied in parallel batch mode fermentations using E. coli BL21(DE3)Gold as a host. After improvement of fed-batch high density fermentation conditions, 153 mg fusion protein corresponding to 51.5mg native proinsulin was obtained per L. Proteins were extracted from the periplasm by osmotic shock treatment. The fusion protein was purified in one step by ecotin affinity chromatography on immobilized trypsinogen. After thrombin cleavage of the fusion protein, the products were separated by Ni-NTA chromatography. Proinsulin was quantified by ELISA and characterized by mass spectrometry. To evaluate the influence of periplasmic proteases, the amount of ecotin-proinsulin was determined in E. coli BL21(DE3)Gold and in a periplasmic protease deficient strain, E. coli SF120.     

Overexpression of protein disulfide isomerase DsbC stabilizes multiple-disulfide-bonded recombinant protein produced and transported to the periplasm in Escherichia coli

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.     

Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.     

Bacterial expression and purification of the Fab fragment of a monoclonal antibody specific for the low-density lipoprotein receptor-binding site of human apolipoprotein E.

We report the bacterial expression and the purification of a monoclonal antibody (mAb) specific for an epitope that coincides with the LDL receptor (LDLr)-binding domain of human apolipoprotein E (apoE). This antibody resembles the LDLr in its primary structure and its specificity for apoE variants. The recombinant Fab (rFab) fragment of mAb 2E8, consisting of the entire light chain and the Fd portion of the heavy chain, was expressed in Escherichia coli and purified to homogeneity. Purification was facilitated by including a five-histidine carboxyl-terminal extension on the Fd chain. A 5- to 10-fold difference in yield of the antibody was observed when the plasmid was expressed in two different strains of E. coli. Typically 2-6 mg of rFab per liter of culture medium was recovered in the periplasm of the TG1 strain and less than 1 mg was recovered in the periplasm of the XL1-Blue strain. Culture temperatures above 35 degrees C or inclusion of sucrose in the medium reduced rFab yields. The 2E8 rFab was indistinguishable from Fab prepared from 2E8 hybridoma-generated IgG with respect to its affinity and fine specificity. We are using this system to express a panel of 2E8 variant Fabs that will be used as probes to establish the structural features responsible for the binding of apoE to the LDLr.     

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.     

ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli.

ClpB is a heat-shock protein from Escherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mM ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When the chaperones were added to the luciferase refolding solutions after 5-25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.     

Bacterial expression and purification of recombinant bovine Fab fragments.

We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.     

Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli

Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permit dsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.     

Simian virus 40 T antigens and J domains: analysis of Hsp40 cochaperone functions in Escherichia coli

The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones. We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function. We found a strong correlation between loss of cochaperone function in E. coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function. We also report the expression of native SV40 virus T antigens in E. coli. Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo. In addition, purified small t antigen can efficiently stimulate E. coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone. Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E. coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart. Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.     

Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.