Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.     

Expression of proteins using the third domain of the Escherichia coli periplasmic-protein TolA as a fusion partner

The third domain of the periplasmic protein TolA from Escherichia coli (TolAIII) was used as a fusion partner in the expression of various proteins from bacteria and eukaryotes. TolAIII is small domain, expressed in high yields as a soluble protein in the cytoplasm of E. coli. Proteins were linked to the C-terminus of TolAIII by a short flexible linker containing sites for endopeptidases. Three different vectors were prepared, containing sites for enterokinase, thrombin or factor Xa. Fusion proteins also contain a His(6)-Ser(2) tag at their N-terminus for easier purification. Up to 90 mg fusion protein per liter bacterial culture was obtained using these vectors. Colicin N R-domain was expressed with this system as a fusion and processed further for functional studies. The yield of final pure R-domain was doubled as compared to the direct expression. The system may prove to be useful in the preparation of other peptides and proteins.     

β-lactamase as a probe of membrane protein assembly and protein export

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.     

Protein secretion in gram-positive bacteria.

Gram-positive bacteria often secrete large amounts of proteins into the surrounding medium. This feature makes them attractive as hosts for the industrial production of extracellular enzymes. Compared to Escherichia coli, relatively little is known about the mechanism of protein secretion in these organisms. However, the recent identification of Bacillus subtilis genes whose gene products are highly homologous to some of the Sec (secretion) proteins of E. coli strongly suggests that important principles of protein translocation across the plasma membrane might be highly conserved. In contrast, the steps following the actual translocation event might be different in Gram-positive and Gram-negative bacteria. The scope of this review is to outline the recent progress that has been made in the elucidation of the secretion pathway in Gram-positive bacteria and to discuss potential applications in strain improvement for the industrial production of extracellular proteins.     

Pattern recognition by TREM-2: binding of anionic ligands

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.     

蛋白A信号肽引导的E.coli外泌高表达异源蛋白

利用葡萄球菌ｐｒｏｔｅｉｎ Ａ信号序列（ＳＰＡ）,我们构建了不同启动子控制的分泌表达质粒。经表达研究,获得了可控性好、表达量高的ＰＬ启动子控制的分泌表达载体。通过菌种的筛选、培养条件和诱导条件的摸索,获得了能将表达产物的绝大部分分泌到培养液中的大肠杆菌高效外泌表达系统,外泌表达量可达１００ｍｇ／Ｌ（菌浓度为１Ａ６００／ｍｌ）以上,如此高的分泌表达量尚未见文献报道。利用该系统成功、高效地外泌表达了ｐ     

Improved expression and purification of the correctly folded response regulator PlnC from lactobacilli

The response regulator PlnC is part of the signal transduction system that plays a key role in the regulation of bacteriocin production in Lactobacillus plantarum C11. In this study, we wanted to express high levels of the response regulator PlnC in a soluble and native form for purification and further studies. The protein was expressed as a fusion protein (fPlnC) containing an N-terminal Flag-tag to facilitate detection and purification. When the fusion gene, fplnC, was expressed in Escherichia coli BL21, nearly all (99%) of the recombinant protein ended up inside inclusion bodies as an incorrectly folded protein. By utilizing two different Gram-positive expression systems (SIP and NICE) in L. plantarum NC8 and Lactobacillus sakei Lb790, the expression of the soluble fPlnC was significantly increased, being 20-40 times more than that in E. coli BL21. Using the N-terminal tag, the expressed protein was purified by immunoprecipitation. By DNA-binding study (EMSA), we demonstrated that the fusion protein purified from the soluble pool was correctly folded as judged by its ability to bind specifically on regulated promoters. Using our approach, we estimate that about 1 mg of fPlnC can be purified from 11 of the bacterial culture.     

Inhibitorily active recombinant human stefin B. Gene synthesis, expression and isolation of an inhibitory active MS-2 pol-stefin B fusion protein and preparation of Des[Met1,2(2)]stefin B

A synthetic gene coding for the human intracellular cysteine proteinase inhibitor, stefin B, was constructed from 13 chemically synthesized oligonucleotides according to the method of Khorana. The gene was inserted into the plasmid vector pTZ, amplified and sequenced. For expression, a temperature-inducible system producing fusion proteins was used. With the vector pEx31A containing the synthetic cystatin B gene, E. coli strain 537 produced a fusion protein of the N-terminal part of bacteriophage MS-2 polymerase and [Met-2Gly-1]stefin B. Lysates of the induced bacteria were inhibitorily active against papain. The fusion protein was expressed in high yield (about 20% of total E. coli proteins) and mostly deposited as inclusion bodies. The unfolded fusion protein was partially purified in the presence of urea. After refolding, approx. 6% of the protein was inhibitorily active against papain, human cathepsin H and B. Des[Met1,2(2)]stefin B was released by cyanogen bromide cleavage of the fusion protein and identified by N-terminal amino-acid sequence analysis. The non-separated cleavage products were also inhibitorily active after refolding. The estimated inhibition constants for the fusion protein and its cleavage products were similar to those reported for natural stefin B.     

Recent advances in recombinant protein expression by Corynebacterium, Brevibacterium, and Streptomyces: from transcription and translation regulation to secretion pathway selection

Gram-positive bacteria are widely used to produce recombinant proteins, amino acids, organic acids, higher alcohols, and polymers. Many proteins have been expressed in Gram-positive hosts such as Corynebacterium, Brevibacterium, and Streptomyces. The favorable and advantageous characteristics (e.g., high secretion capacity and efficient production of metabolic products) of these species have increased the biotechnological applications of bacteria. However, owing to multiplicity from genes encoding the proteins and expression hosts, the expression of recombinant proteins is limited in Gram-positive bacteria. Because there is a very recent review about protein expression in Bacillus subtilis, here we summarize recent strategies for efficient expression of recombinant proteins in the other three typical Gram-positive bacteria (Corynebacterium, Brevibacterium, and Streptomyces) and discuss future prospects. We hope that this review will contribute to the development of recombinant protein expression in Corynebacterium, Brevibacterium, and Streptomyces.     

Systematic analysis of fusion and affinity tags using human aspartyl-tRNA synthetase expressed in E. coli

Fusion and affinity tags are popular tools for the expression of mammalian proteins in bacteria. To facilitate the selection of expression approaches, a systematic comparison was performed. We cloned, sequenced, and expressed in Escherichia coli ubiquitin- and SUMO-hDRS fusion proteins with biotin- or 6xHis-tags. The tagging of hDRS with ubiquitin or SUMO was necessary to express properly folded and biologically active enzyme. Similar enhancement of hDRS activity was obtained by fusion to ubiquitin or SUMO. Ubiquitin, SUMO, biotin, and hexahistidine tags did not appreciably interfere with hDRS activity. Fusion proteins were specifically cleaved without altering the N-terminal of hDRS. After cleavage hDRS remained soluble and active with a specific activity comparable to that of the fused protein. Similar activity was observed with biotin- and 6xHis-tagging of hDRS. Higher purity but significantly lower yields of hDRS were obtained using biotin-tagging. Overall we demonstrated ubiquitin and SUMO fusion proteins similarly enhanced the proper folding of hDRS expressed in E. coli. In comparison to previous expressions of hDRS as a GST fusion, ubiquitin, and SUMO fusions provided higher yields and easier purification and cleavage.     

Soluble fusion expression and characterization of human beta-defensin 3 using a novel approach

Human β-defensin 3 (DEFB103) is a recently identified small cysteine-rich cationic peptide expressed ubiquitously upon local microbial invasion. A number of accumulating evidences indicate that this peptide is involved in many biological processes, including microbicidal activities, chemoattraction, and immunomodulation. In this article, we describe a novel approach through which we performed the expression and purification of the recombinant DEFB103 peptide in Escherichia coli (E. coli) based on the pTWIN1 expression system. This approach does not introduce any extra residues to the peptide product, and also eliminates the requirement of removing the fusion tag by exogenous proteases. A high yield of 112 mg of soluble fusion DEFB103 was obtained in 1 liter of Luria-Bertani (LB) medium. By one-step affinity chromatography and on-column, auto-cleavage of the fusion tag, the mature DEFB103 peptide was produced with a yield of 30 mg/L LB. The purified DEFB103 peptide demonstrated strong antimicrobial activities against E. coli, S. aureus and C. albicans, which were representatives of Gram-negative and Gram-positive bacteria and fungi, respectively. Using this novel approach, we have successfully expressed and purified several human defensins with significant bioactivities. Our work may be helpful for structural and functional studies of other human defensins, and also for the production of human defensins.     

重组人干扰素α-2b在大肠杆菌中分泌表达

人干扰素α-2b原始基因在重组原核工程菌中表达量偏低,所以我们在不改变干扰素原有氨基酸组成的前提下,根据大肠杆菌密码子偏爱性使用定向突变技术对huIFNα-2b基因进行点突变。将大肠杆菌STⅡ信号肽基因与突变后huIFNα-2b基因融合并于信号肽5′端和huIFNα-2b基因3′端引入合适的酶切位点。融合基因克隆至载体pCSE,pET-22b和pPAK4L中,此3种载体分别含有组成型启动子、T7启动子和phoA启动子。融合基因在载体pCSE中表达量很低,其中约有50%的目标蛋白能够成功实现分泌。在E.coliBL21中,pET-22b经过IPTG诱导可以实现huIFNα-2b的高表达,但STⅡ信号肽不能被有效切除。含有phoA启动子的载体pPAK4L其在E.coliW3110中可以实现huIFNα-2b较高水平的分泌表达,经过低磷诱导其表达量最高可至20μg/mL(A550)菌液,约有30%的目标蛋白质信号肽能够被成功切除并分泌到胞间质中。     

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.     

大肠杆菌YggG与结合蛋白Era的相互作用研究

Era是细菌生长必须的一高度保守的GTPase。yggG是从大肠杆菌全基因组文库中钓取并克隆的Era结合蛋白基因,进一步的研究表明该基因在大肠杆菌中的表达与环境应激相关,提示yggG基因产物参与细菌的应激调控。为了阐明YggG蛋白与Era蛋白间的相互关系,利用所构建的双启动子表达载体pDH2-YggG-Ptac-Era在同一细胞中同时表达YggG与Era蛋白,并通过免疫共沉淀实验检测细菌裂解产物YggG与Era蛋白间的相互作用;在此基础上,构建并表达纯化了GST融合的Era蛋白氨基端截短肽和Era羧基端截短肽,通过GST Pull-down检测了Era不同功能区域与YggG蛋白间的相互作用。结果显示, Era/YggG 复合物仅存在于同时过表达Era和YggG蛋白的细菌细胞内,不诱导Era或者不诱导YggG蛋白过表达,均检测不到Era/YggG 复合物存在;纯化的GST不能Pull-down YggG蛋白,而纯化的GST融合的Era蛋白、Era氨基端截短肽及Era羧基端截短肽均可以Pull-down YggG蛋白;GST融合Era氨基端截短肽和GST融合的Era蛋白对YggG蛋白结合作用明显高于GST融合的Era蛋白羧基端截短肽。上述结果说明,YggG是一大肠杆菌Era结合蛋白,YggG与Era的氨基端和羧基端的结合活性存在差异。     

Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes

A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.     

Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.     

超抗原SEA与抗黑色素瘤ScFv融合基因的构建及表达

采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肽的金黄色葡萄球菌肠毒素A基因进行融合,并将融合基因克隆于pET28a表达载体上,转化大肠杆菌BL21(DE3)。用NiNTA系统对表达产物进行分离、纯化。MTT法检测融合蛋白对黑色素瘤细胞的体外抑制率。结果表明6HisScFvSEA融合蛋白可在E.coli BL21(DE3)中稳定表达,表达量占菌体蛋白的30%,主要以包涵体的形式存在。融合蛋白可通过激活效应细胞对表达相关抗原的黑色素瘤细胞发挥抑制作用。     

Codon preference optimization increases heterologous PEDF expression

Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5' thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes, respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ～3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ～4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ～3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at -3 to +50 of the Trx site of both fusion genes were -3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.     

鸡源抗菌肽Fowlicidin-2在大肠杆菌中的 重组表达及其生物学活性鉴定

【目的】对抗菌肽Fowlicidin-2基因进行克隆与表达,并鉴定其生物学活性。【方法】根据抗菌肽Fowlicidin-2氨基酸序列,依照大肠杆菌(E.coli)密码子的偏爱性,人工设计合成其编码基因。与质粒pET-32a连接,构建重组表达载体,转化表达宿主菌E.coliBL21(DE3),IPTG诱导表达,融合蛋白经溴化氰裂解后进行纯化,测定重组抗菌肽的抑菌活性。【结果】Fowlicidin-2融合蛋白以包涵体形式表达,经溴化氰裂解后,成功释放出Fowlicidin-2,获得的重组Fowlicidin-2对革兰氏阳性菌和革兰氏阴性菌均有明显的抑菌效果。【结论】实现了抗菌肽Fowlicidin-2的重组表达,为抗菌肽的重组量化制备提供了理论基础与技术手段。     

Nitrogenase MoFe protein subunits from Klebsiella pneumoniae expressed in foreign hosts. Characteristics and interactions

The expression of selected nitrogen fixation (nif) genes from Klebsiella pneumoniae in foreign hosts provides an approach to determine the pathway, minimal genetic requirements, and host dependence of nitrogenase assembly. In this study, we investigated the assembly of the alpha 2 beta 2 MoFe protein, responsible for substrate binding and reduction, by introducing nifD and nifK (encoding respectively, the alpha and beta subunits) into Escherichia coli and the yeast Saccharomyces cerevisiae. In E. coli, both genes were expressed from the nifHDKY operon; in yeast, the genes, separately fused to the yeast ADH1 promoter, were introduced on two different plasmids. Denaturing immunoblot analyses demonstrated the presence of significant amounts of NifD and NifK in both hosts. In E. coli, the level or perhaps modification of NifD depended on the growth medium of the bacteria. Nondenaturing, anaerobic immunoblot assays revealed in E. coli, nif-specific antigens of lower electrophoretic mobility than Kp1, which may represent assembly intermediates. In yeast, no putative assembled products were evident, and the predominant antigens corresponded to the monomeric forms of the polypeptides. These results indicate that, unlike NifH, the Fe protein subunit (Berman, J., Gershoni, J. M., and Zamir, A. (1985) J. Biol. Chem. 260, 5240-5243), NifD and NifK are insufficient for the assembly of an electrophoretically Kp1-like structure. Homodimerization of nifK and probably of nifD primary gene products does not appear to occur spontaneously and hence is unlikely to represent the initial step in the assembly. The difference between the two hosts suggests that the cellular environment or mode of expression could affect the interaction between the two subunits.