抗菌肽在大肠杆菌中的融合表达

抗菌肽作为新一代抗生素的潜在应用价值使其备受关注,大量高纯度的抗菌肽是开展基础及临床实验的关键。天然来源的抗菌肽资源有限、纯化困难,化学合成抗菌肽成本高、活性不稳定,因此通过基因重组表达得到大量抗菌肽是低成本、高效益的方法。目前采用大肠杆菌表达系统获得抗菌肽已成为研究者的首选,通常以形成融合蛋白的方式表达,这不仅可避免抗菌肽对宿主的杀伤作用,也保护了抗菌肽免受蛋白酶降解。文章结合课题组的研究工作,综述了近年来抗菌肽在大肠杆菌中表达的融合载体、融合蛋白的裂解方法及表达条件优化的研究进展。     

Recombinant expression of indolicidin concatamers in Escherichia coli

Antimicrobial peptides are part of the innate immune system of vertebrates and invertebrates. They are active against gram-negative and gram-positive bacteria, fungi, and protozoa. Currently, most antimicrobial peptides are extracted from host organisms or produced by solid-phase peptide synthesis. Recombinant protein expression in Escherichia coli is a tool for greater production yields at a decreased cost and reduces the use of hazardous materials. We have constructed a concatamer of indolicidin and successfully expressed a fusion product with thioredoxin in E. coli BL21DE3. Codons for methionine residues flanking individual indolicidin genes were incorporated for cyanogen bromide cleavage of the fusion protein and liberation of active monomeric indolicidin. Peptide yields of 150 μg/l monomeric indolicidin were achieved in this first report of recombinant production of indolicidin with demonstrated antimicrobial activity.     

Use of magnetic carboxyl beads to purify a cationic peptide in a batch system

Antimicrobial peptides (AMPs) are cationic molecules that are good leads for new antiinfective drugs. To obtain sufficient amounts, recombinant AMPs are generally produced as fusion proteins in Escherichia coli. Fusion partners facilitate purification of recombinant proteins. Fusion proteins are then cleaved by specific proteases, and cationic peptides are purified by size exclusion chromatography or ion exchange chromatography, neither of which is easily applicable to small volumes of diluted peptide samples. We developed a small-scale system that is easily adaptable for high-throughput screening and uses carboxyl magnetic beads to purify a cationic peptide from its fusion partner.     

杂合抗菌肽牛乳铁蛋白素-天蚕素在大肠杆菌中的高效表达及其活性鉴定

【目的】菌株耐药性问题日益突出,研制新型安全高效的抗菌药物成为目前的研究热点之一。抗菌肽具有多种优良特性,高活性抗菌肽的开发及其重组表达对解决菌株耐药性问题具有重要意义。【方法】根据牛乳铁蛋白素与天蚕素的结构,设计一种新型的杂合抗菌肽牛乳铁蛋白素-天蚕素(LfcinB-Cecropin),根据Escherichia coli密码子偏爱性合成其编码基因,利用同尾酶法构建含有不同LfcinB-Cecropin基因片段拷贝数的重组表达载体,转化到E.coli BL21(DE3)进行重组表达。【结果】经IPTG诱导,LfcinB-Cecropin融合蛋白成功获得表达。经超声破碎、包涵体纯化、甲酸裂解后,获得具有明显抑菌活性的杂合肽LfcinB-Cecropin。【结论】获得一种高活性的新型抗菌肽LfcinB-Cecropin,并实现了在E.coli中的高效重组表达。     

利用ELP自断裂标签在大肠杆菌中生产抗菌肽Oxysterlin 1

抗菌肽是目前最有希望的抗生素替代品,但是使用重组技术生产抗菌肽的策略大多步骤烦琐且价格昂贵,不利于抗菌肽的规模化生产。Oxysterlin 1是一种新型的天蚕素抗菌肽,主要对革兰氏阴性菌有抗菌活性,具有较低的细胞毒性。文中利用一种简单经济的方法在大肠杆菌中实现Oxysterlin 1的表达和纯化。将Oxysterlin 1基因克隆到含有弹性蛋白样多肽Elastin-like polypeptide (ELP) 和蛋白质内含肽 (Intein) 的载体中,构建重组表达质粒pET-ELP-I-Oxysterlin 1。重组蛋白在大肠杆菌中主要以可溶性形式表达,进而通过简单的盐析和pH改变便可对目标小肽进行纯化。最终得到的Oxysterlin 1的产量约为1.2 mg/L,抑菌试验显示出预期活性,为抗菌肽的规模化生产及深入研究其抑菌机理奠定基础。     

迟眼蕈蚊抗菌肽BhSGAMP-1-S在大肠杆菌中的优化表达及其活性分析(英文)

【目的】BhSGAMP-1 是迟眼蕈蚊唾液腺抗菌肽,为了能够更好的了解其分子特性,我们将其表达、纯化并进行了活性测定。【方法】依据大肠杆菌稀有密码子设计并合成了抗菌肽基因 BhSGAMP-1-S,以 pMAL-c2X 作为表达载体在大肠杆菌 TB1 中进行融合表达,融合蛋白通过麦芽糖亲和层析柱进行纯化,获得的融合蛋白经肠激酶切割后,混合物通过分子筛凝胶层析和反相高效液相色谱来获得单体重组抗菌肽 BhSGAMP-1-S,对获得的抗菌肽进行活性测定。【结果】在最优的表达条件下融合蛋白以可溶的形式表达,100 mL 诱导菌液经多步纯化后可得 0. 38 mg 的重组抗菌肽 BhSGAMP-1-S,抑菌活性测定表明所获得的抗菌肽对部分测试革兰氏阳性细菌、革兰氏阴性细菌和真菌有较强的抑菌活性。【结论】本研究第一次成功的在大肠杆菌中诱导表达了修饰合成的抗菌肽 BhSGAMP-1-S,纯化后的抗菌肽具有很好的抑菌活性,这为进一步研究和应用奠定了基础。     

A fusion protein system for the recombinant production of short disulfide-containing peptides

A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.     

Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners

It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides.     

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His(6) fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.     

黄鳍金枪鱼抗菌肽YFGAP在大肠杆菌中融合表达及其活性检测

【目的】抗菌肽YFGAP由32个氨基酸组成,分子量为3.4 kD,对革兰氏阳性菌(G+)和革兰氏阴性菌(G?)表现出强效的抑制作用,不具有溶血活性。在大肠杆菌中表达抗菌肽YFGAP,分离纯化抗菌肽并鉴定其生物学活性。【方法】化学合成EK-YFGAP和L-EK-YFGAP基因序列,构建表达载体pET22b-ELP20-EK-YFGAP、pET22b-ELP40-EK-YFGAP和pET22b-ELP40-L-EK- YFGAP,分别转化至大肠杆菌BL21(DE3)中诱导表达,可逆相变循环纯化融合蛋白。肠激酶酶切,经Vivaspin Turbo纯化柱纯化,测定重组抗菌肽的抑菌活性和溶血活性。【结果】纯化出两种融合蛋白ELP40-EK-YFGAP和ELP40-L-EK-YFGAP,肠激酶酶切纯化后获得重组抗菌肽YFGAP,对4种病原菌均有抑制效果,溶血活性较低。【结论】以ELPs作为非色谱纯化标签,实现了抗菌肽YFGAP的融合表达,具有操作简单、成本低、易于扩大的优势,为重组抗菌肽的量化制备及应用提供了理论基础和技术支持。     

Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.     

Antimicrobial activity of recombinant Pg-AMP1, a glycine-rich peptide from guava seeds

Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98kDa as a monomer and 13.96kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.     

Production of recombinant peptides as fusions with SUMO

Recombinant production of non-native peptides requires using protein fusion technology to prevent peptide degradation by host-cell proteases. In this work, we have used SUMO protein as a fusion partner for the production of difficult-to-express, antimicrobial, self-assembling and amyloidogenic peptides using Escherichia coli. SUMO-peptide fusions were expressed as intracellular products by utilizing pET based expression vectors constructed by Life Sensors Inc., USA. Histidine tagged SUMO-peptide fusions were purified using Ni-NTA affinity chromatography. Complete (100%) cleavage of the SUMO-peptide fusion was achieved using SUMO protease-1. Our findings demonstrate that SUMO fusion technology is a promising alternative for production of peptides in E. coli. The key advantage of this technology is that the enzymatic activity of SUMO protease-1 is specific and efficient leading to inexpensive costs for cleaving the peptide fusion when compared with other fusion systems.     

High-level expression of antimicrobial peptide mediated by a fusion partner reinforcing formation of inclusion bodies

A gene expression system for antimicrobial peptides, which could be effectively used for various studies or applications of the antimicrobial peptides, has been developed. To avoid the harmful effects on an expression host, Escherichia coli, the antimicrobial peptides were expressed as fusion proteins with a polypeptide F4, which is a truncated PurF fragment that highly tends to form inclusion bodies. Seven different kinds of antimicrobial peptides have been successfully expressed by this expression system and the resulting expression level of fusion proteins reached up to 30% of total cell proteins. To confirm the identity of the recombinant peptide, MSI-344 was selected as a model peptide and purified to homogeneity, and we could obtain the recombinant MSI-344 of a high purity and with a good yield, which was identical to the authentic peptide in the aspects of the chemical and antimicrobial properties. These results show that the neutral fusion partner, which reinforces the formation of inclusion bodies, could mediate a high-level expression of the antimicrobial peptides.     

破菌时可自动降解宿主核酸的大肠杆菌BL21(DE3)的lpxM突变株构建

研究利用Red同源重组技术对常用大肠杆菌表达宿主菌BL21(DE3)进行改良, 构建破菌时可自动降解宿主核酸的大肠杆菌表达宿主菌, 该菌株可望有助于解决因破菌时宿主菌染色体核酸释放给后续纯化重组蛋白工作带来的困难。将N端连有OmpA的信号肽的S. aureus nucleaseB(nucB)表达框整合至E. coli BL21(DE3)的lpxM位点, 改造后菌株(称为BLN)经诱导能表达nucB、并分泌至周质空间, 这样可使宿主核酸免受该酶“毒性”影响, 菌体裂解后, nucB释放,能自动降解宿主核酸。BLN菌体生长状态以及表达外源重组蛋白的能力与出发菌基本一致。     

Recombinant production of cathelicidin-derived antimicrobial peptides in Escherichia coli using an inducible autocleaving enzyme tag

Antimicrobial peptides (AMPs), such as the linear amphipathic cathelicidins, are produced widely in the natural world and are active against a broad range of pathogenic microorganisms. Their potential as a new range of antibiotics has prompted numerous studies of AMP structure and function. Most such studies are performed with chemically synthesised peptides, but a simple and rapid biosynthetic route would offer a more cost-effective alternative for the production of AMPs and analysis of their structure/function relationships. The cysteine protease domain (CPD) from Vibrio cholerae MARTX toxin possesses an autocleaving ability that is inducible by inositol hexakisphosphate (IP(6)). When coupled with a hexa-histidine tag and fused to the C-terminus of an AMP, this AMP-CPD fusion may be expressed in Escherichia coli and purified using immobilized metal affinity chromatography. A brief on-column induction of cleavage liberates the AMP, and subsequent polishing using hydrophobic interaction resin allows for purification of the peptide within a day. We used this system to express and purify several 18-residue cathelicidin variants and tested their activity on E. coli, Pseudomonas putida, Bacillus subtilis and Candida albicans. This approach to linear AMP production may aid rapid construction and purification of structural variants for subsequent functional analysis.     

Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli

Insect cecropins are small basic polypeptides synthesized in fat body and hemocytes in response to bacterial infections or hypodermic injuries. To explore a new approach for high expression of soluble cecropin in Escherichia coli cells, we fused the sequence encoding Musca domestica mature cecropin (named Mdmcec) in-frame to thioredoxin (TRX) gene to construct an expression vector pTRX-6His-Mdmcec. An enterokinase cleavage site was introduced between the 6xHis-tag and Mdmcec to facilitate final release of the recombinant Mdmcec. The fusion protein TRX-6His-Mdmcec was purified successfully by HisTrap HP affinity column and a high yield of 48.0mg purified fusion protein was obtained from 1L culture. Recombinant Mdmcec was readily obtained by enterokinase cleavage of the fusion protein followed by HPLC chromatography, and 11.2mg pure active recombinant Mdmcec was obtained from 1L E. coli culture. The molecular mass of recombinant Mdmcec determined by electrospray ionization-mass spectrometry (ESI-MS) is identical to that of native cecropin. Analysis of recombinant Mdmcec by circular dichroism (CD) indicated that recombinant Mdmcec contained predominantly alpha-helix with some random coil. Antimicrobial activity assays demonstrated that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria. The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies.     

Advanced genetic strategies for recombinant protein expression in Escherichia coli

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology. Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E. coli cytoplasm. Here we review the current most important strategies for recombinant expression in E. coli. Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology. The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.     

抗菌肽Pexiganan和IB-367的基因克隆及表达

目的:为开发新的抗菌药,通过基因工程手段获得能够表达抗菌肽Pexiganan和IB-367的工程菌株。方法:人工合成2种抗菌肽Pexiganan和IB-367基因,构建相应的GST融合表达载体,转化大肠杆菌后筛选阳性克隆,通过SDS-PAGE和Western印迹验证目的蛋白的表达。结果:获得了2株分别表达Pexiganan-GST融合蛋白和IB-367-GST融合蛋白的工程菌株。结论:通过基因工程方法,可以融合蛋白的形式获得小分子多肽Pexiganan和IB-367。