Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy.

A dual affinity fusion approach has been used to study the expression and secretion of labile recombinant proteins in Escherichia coli. Here we show that three small eukaryotic proteins (human proinsulin, a thioredoxin homologous domain of rat protein disulfide isomerase, and the extracellular domain of the alpha 1.2-chain of a human T-cell receptor) are stabilized in vivo using a dual affinity fusion strategy, where the gene encoding the desired product is fused between two genes encoding two different affinity domains. Relatively high yields of full-length product were obtained for all three proteins as compared to when fused to a single fusion partner. Despite the use of a signal peptide, significant amounts of the disulfide protein isomerase and T-cell receptor gene products were maintained in the cytoplasm, while the proinsulin fusion was efficiently secreted to the periplasm. Interestingly, the E. coli heat shock proteins DnaK and GroEL were associated with the fusion proteins isolated from the cytoplasm.     

The extracellular domain of the human erythropoietin receptor: expression as a fusion protein in Escherichia coli, purification, and biological properties

We developed an efficient production system of the soluble extracellular domain of the human erythropoietin receptor (sEPO-R) and characterized the binding of erythropoietin (EPO) with the purified recombinant protein. The sEPO-R, fused to the maltose binding protein (MBP), was expressed as a soluble protein in the periplasm of Escherichia coli (E. coli) and did not accumulate in inclusion bodies. After lysis of the bacteria by an osmotic shock, the fusion protein was purified by affinity chromatography on amylose followed by size exclusion chromatography (SEC). Specific binding of 125I-labelled EPO to the sEPO-R was demonstrated by competitive and saturation binding assays. A single affinity class (Kd = 0.25 nM) of the binding site was evident by Scatchard analysis. This value is similar to the Kd observed between EPO and the EPO-R of high affinity present on human erythroid progenitors. The complex has a molecular size corresponding to a 1:1 complex of EPO and the fusion protein.     

Staphylococcal protein A as a fusion partner directs secretion of the e1alpha and e1beta subunits of pea mitochondrial pyruvate dehydrogenase by Bacillus subtilis

Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1alpha and E1beta subunits of Pisum sativum mitochondrial pyruvate dehydrogenase from Bacillus subtilis. These proteins were not exported when the signal peptide from levansucrase (SacBSP) was fused to their N-termini. Both SacBSP-E1alpha and SacBSP-E1beta fusion proteins were insoluble in the cytoplasm. However, when the SPA open-reading frame was inserted between SacBSP and E1alpha or E1beta, corresponding fusion proteins were secreted from the cells. The first (E) IgG-binding domain of SPA was sufficient to direct low level secretion of both fusion proteins (SacBSP-E-E1alpha and SacBSP-E-E1beta). Adding the second (D) IgG-binding domain improved extracellular protein yields 3- to 4-fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins. All constructs were based on the pUB110-derived multicopy plasmid pWB705. Separate B. subtilis strains transformed with SacBSP-E-E1alpha-His(6) or SacBSP-E1beta were cocultivated in the presence of Ni-NTA agarose. The native pyruvate dehydrogenase alpha2beta2 structure was bound to the affinity matrix, demonstrating assembly after secretion. The use of SPA as a fusion partner during expression of heterologous proteins by B. subtilis provides the basis of a versatile system that can be used to study both secretion and protein:protein interactions.     

Substitution of surface-exposed framework residues alters secretion of recombinant fusion protein Fv/tumor necrosis factor in Escherichia coli

Substitution of hydrophobic residues with hydrophilic ones at surface-exposed positions may influence the yield of antibody fragment expression in Escherichia coli by reducing its aggregation potential. We introduced such substitutions at 8 surface-exposed framework region residues of a fusion protein Fv/Tumor Necrosis Factor, which resulted in the expression of 10 mutant fusion proteins (Mut 1-10) in E. coli. Our results showed that expression levels of Mut 1-3, with mutations of A9S, T18K, and G41D, respectively, decreased by 4- to 8-fold, whereas expression levels of Mut 4, 9, and 10, with mutations of S60D/S70D, T28D, and G65D, respectively, were not affected. In contrast, mutation of F83A at light-chain residue 83, which is usually buried at the variable/constant domain interface of antibody molecules but becomes surface-exposed in recombinant Fv molecules, increased the expression level of Mut 5 by 4-fold. Our results suggest that this important substitution of a hydrophobic residue with a hydrophilic one may also be applied to increase the secretion of other recombinant Fv molecules in E. coli periplasm.     

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.     

掺入大肠杆菌膜中的磷脂酰胆碱(PC)影响青霉素b-内酰胺酶的分泌

[目的]为了研究磷脂酰胆碱(PC)在原核生物细胞中的生物学作用,探讨PC对细菌膜系统的功能的影响.[方法]使用ptac 85质粒作载体,将螺旋菌pcs基因导入E.coli Top10细胞构建了E.coli Fop10 pcs 菌株,并在特定的条件下培养细菌,使细菌膜磷脂中合成30%左右的磷脂酰胆碱.然后再使用抗生素抗性分析、β-内酰胺酶的酶活测定以及Western blot杂交技术,分析质粒编码的β-内酰胺酶从细胞质到细胞问质的分泌情况.[结果]抗生素抗性分析发现,高浓度的氨苄青霉素抑制E.coliTop10 pcs 细菌的生长的氨苄青霉素剂量低于对照组,其半致死剂量IC50在700～800μg/mL之间.酶活检测显示E.coli Top10 pcs 细菌周质内β-内酰胺酶的酶活性只有对照菌株的1,5,Western blot进一步分析发现周质内β-内酰胺酶的含量也为对照菌株的1/5.由此可见,周质内低含量的β-内酰胺酶是导致E.coli Top10pcs 细菌氨苄青霉素抗性降低的原因.[结论]掺入细菌膜磷脂双分子层的PC影响p.内酰胺酶通过Sec转运途径从细胞质分泌到细菌周质空间内,提示细菌磷脂酰胆碱可能在调节蛋白转运和分泌方面起着重要的作用.     

Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain

The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis. Single-chain antibody fragments (scAb) are scFv with a human kappa light chain constant (HuCkappa) domain attached C-terminally and share similar problems of expression. By fusing the E. coli maltose-binding protein (mbp) gene either 3' or 5' to a scAb specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion. The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely. Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scAb in the periplasmic compartment, indicating that periplasmic scAb solubility is unrelated to propensity to cause growth arrest. IMAC purification of scAb and mbp-scAb proteins followed by liquid competition ELISA revealed the IC(50) for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding. The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCkappa domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion. The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCkappa domain fusion were extended to a second scFv specific for the herbicide diuron. Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCkappa) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E. coli periplasm.     

Analysis of factors affecting the periplasmic production of recombinant proteins in Escherichia coli

Five fusion proteins between Z domains derived from Staphylococcal Protein A and Green Fluorescent Protein or Human Proinsulin were produced on the periplasm of Escherichia coli. The effects of the molecular weight and amino acid composition of the translocated peptide, culture medium composition, and growth phase of the bacterial culture were analyzed regarding the expression and periplasmic secretion of the recombinant proteins. It was found that secretion was not affected by the size of the translocated peptide (17-42 kDa) and that the highest periplasmic production values were obtained on the exponential phase of growth. Moreover, the highest periplasmic values were obtained in minimal medium, showing the relevance of the culture medium composition on secretion. In silico prediction analysis suggested that with respect to the five proteins used in this study, those that are prone to form alpha-helix structures are more translocated to the periplasm.     

Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization,yield and activity

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.     

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane.

The plant pathogenic enterobacterium Erwinia chrysanthemi EC16 secretes several extracellular, plant cell wall-degrading enzymes, including pectate lyase isozyme PelE. Secretion kinetics of 35S-labeled PelE indicated that the precursor of PelE was rapidly processed by the removal of the amino-terminal signal peptide and that the resulting mature PelE remained cell bound for less than 60 s before being secreted to the bacterial medium. PelE-PhoA (alkaline phosphatase) hybrid proteins generated in vivo by TnphoA insertions were mostly localized in the periplasm of E. chrysanthemi, and one hybrid protein was observed to be associated with the outer membrane of E. chrysanthemi in an out gene-dependent manner. A gene fusion resulting in the substitution of the beta-lactamase signal peptide for the first six amino acids of the PelE signal peptide did not prevent processing or secretion of PelE in E. chrysanthemi. When pelE was overexpressed, mature PelE protein accumulated in the periplasm rather than the cytoplasm in cells of E. chrysanthemi and Escherichia coli MC4100 (pCPP2006), which harbors a functional cluster of E. chrysanthemi out genes. Removal of the signal peptide from pre-PelE was SecA dependent in E. coli MM52 even in the presence of the out gene cluster. These data indicate that the extracellular secretion of pectic enzymes by E. chrysanthemi is an extension of the Sec-dependent pathway for general export of proteins across the bacterial inner membrane.     

Tolerance of Escherichia coli beta-galactosidase C-terminus to different-sized fusions.

The tolerance of the beta-galactosidase C-terminus to foreign protein fusions has been explored by using different-sized derivatives of the chimeric protein LACVP1. While the molecular mass of the partner domain shows a minor influence on protein toxicity for the producing E. coli cells, it dramatically affects the proteolytic susceptibility of the whole fusion. Surprisingly, the observed structural modulation of proteolysis is not an all-or-nothing process, but it exhibits a continuous effect concomitantly with the length of the fusion. The conformational effects caused by increasingly sized partners seem to progressively expose cryptic protease target sites, initiating a proteolytic cascade that dramatically reduces the yield of the recombinant protein.     

A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli

A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.     

Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli

LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli. Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway. Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm. Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion. Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E. coli and argue against a role for trigger factor in pathway discrimination.     

Facilitating the formation of disulfide bonds in the Escherichia coli periplasm via coexpression of yeast protein disulfide isomerase

Sacchromyces cerevisiae protein disulfide isomerase (yPDI) was expressed in the E. coli periplasm by using plasmids encoding the OmpA-yPDI-(His)(6) fusion gene under the control of the araBAD, trc, or T7 promoter. The expression levels of yeast PDI under these promoters were compared. Our results showed that yeast PDI expressed into the periplasm could catalyze the formation of disulfide bonds in alkaline phosphatase, restoring the phoA(+) phenotype in dsbA(-) mutants. The yeast PDI was purified from the Escherichia coli periplasm and shown to exhibit catalytic properties comparable to those of the rat enzyme with reduced RNase as substrate. In vivo, coexpression of the yeast PDI increased the yield of bovine pancreatic trypsin inhibitor (BPTI) in E. coli by 2-fold, similar to the effect seen previously with the coexpression of the rat enzyme. However yeast PDI was more effective than rat PDI in facilitating the expression of active tissue plasminogen activator (tPA). These results point to differences in the substrate specificity of various PDI enzymes, at least in the context of the E. coli periplasm.     

Stable periplasmic secretion intermediate in the general secretory pathway of Escherichia coli.

The secretion of the Klebsiella oxytoca cell surface lipoprotein pullulanase involves translocation across the cytoplasmic and outer membranes of the Gram-negative bacterial cell envelope. A variant of pullulanase was created by fusing the signal peptide-encoding 5' region of the Escherichia coli gene for periplasmic MalE protein to the 3' end of the pulA gene encoding almost the entire mature part of pullulanase. When produced in E. coli carrying the malE-pulA gene fusion on a high copy number plasmid and the complete set of genes specifically required for pullulanase secretion on a second plasmid, the hybrid protein differed from wild-type pullulanase as follows: (i) it was not fatty-acylated; (ii) it was apparently processed by LepB signal peptidase rather than by LspA lipoprotein signal peptidase; (iii) it was released into the periplasm and was only slowly transported across the outer membrane, and (iv) it was released directly into the medium rather than via the usual surface-anchored intermediate. The hybrid protein was secreted more rapidly when malE-pulA was expressed from a low copy number plasmid. The two steps in the secretion pathway could be totally uncoupled by expressing first the malE-pulA gene fusion and then the cognate secretion genes. These results show that fatty-acylation of wild-type PulA is not essential for secretion but may improve its efficiency when large amounts of the protein are produced, that the two steps in secretion can occur quite independently and that the periplasmic intermediate can persist for long periods under certain circumstances.     

Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB

The use of the uspA and uspB promoters (universal stress promoters) for heterologous protein production in Escherichia coli is described. Best results were obtained with a moderate copy number vector (15-60 copies) bearing the uspA promoter, reaching 4.6 mg/g dry cell weight (DCW) of ZZ-proinsulin secreted to the periplasm and 1.9 mg/g DCW secreted to the culture medium. These values are about 1.7-fold higher than those previously reported with the same ZZ fusion tag and the SpA leader peptide showing that these stress promoters are potentially valuable for recombinant protein secretion in E. coli. It is further demonstrated that the use of M9 minimal medium is advantageous for protein secretion as compared to LB rich medium.     

A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli.

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.     

TIG-Fc真核表达载体的构建及其对自然杀伤细胞功能的影响

T细胞免疫球蛋白和免疫受体酪氨酸抑制性基序结构域\[T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain,TIGIT\]是一种新型的免疫抑制性受体,在机体的免疫调节网络中扮演着重要角色。为了进一步探究TIGIT对自然杀伤（natural killer,NK）细胞的免疫调节功能,构建了融合蛋白TIGIT胞外段（即TIGIT第1~135位氨基酸,以保留功能结构域IgV区,此胞外段简称为TIG）-人免疫球蛋白G3（immunoglobulin G3,IgG3）Fc段的真核表达载体,并对TIG-Fc融合蛋白的表达及其对NK-92细胞功能的影响进行了初步研究。利用分子生物学方法,将携带人TIG与人IgG3 Fc段的基因融合,然后插入真核表达载体pcDNA3.1(-)中,以构建重组质粒pcDNA3.1(-)-TIG-Fc。随后将重组质粒转染至人胚肾细胞HEK-293T中,通过流式细胞仪和Western blot检测TIG-Fc融合蛋白在293T细胞中的表达;再将重组质粒转染至NK-92细胞中,通过WST-1检测TIG-Fc融合蛋白对NK-92细胞增殖的影响,并利用ELISA法检测TIG-Fc融合蛋白对NK-92细胞分泌IFN-γ的水平的影响。结果成功构建了人TIG与人IgG3 Fc融合表达的真核表达载体,且TIG-Fc融合蛋白的表达能够显著抑制NK-92细胞的增殖和分泌IFN-γ的能力（P<0.05）,为后期TIGIT的功能研究奠定了重要基础。     

Enhancement of the efficiency of secretion of heterologous lipase in Escherichia coli by directed evolution of the ABC transporter system

The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.