重组巴曲酶在毕赤酵母中的高效表达

以毕赤酵母为表达系统,建立生产重组巴曲酶的技术工艺路线。通过递归式PCR的方法,人工合成了巴曲酶基因,将其插入pPIC9表达质粒中,转化至毕赤酵母GS115(his4),筛选出的表达株经甲醇诱导,表达了重组巴曲酶,并得以纯化。从每升发酵液中可纯化得到10mg重组巴曲酶,其比活为238NIHunits/mg,分子量为30.55kD。重组巴曲酶在体外可使纤维蛋白凝固,在体内缩短小鼠出血时间。为开发重组的蛇毒类凝血酶止血剂打下了基础。     

人精氨酸酶在毕赤酵母的高效表达、纯化和活性研究

目的：人精氨酸酶（Arginase, Arg）的基因arg在毕赤酵母高效分泌表达,建立相应纯化工艺路线,研究重组人精氨酸酶的活性。方法：将人精氨酸酶基因arg按正确的阅读框架插入到毕赤酵母表达载体pPIC9α信号肽基因后,构建得到重组毕赤酵母表达质粒。转化毕赤酵母GS115筛选高表达菌株。结果：成功构建了酵母表达载体pPIC-Arg,转化毕赤酵母GS115后筛选到分泌表达目的蛋白Arg的菌株,目标蛋白可以分泌到培养基中。经过膜过滤和凝胶过滤层析对培养基上清进行纯化,即可获得纯度达到95%的活性产物。活性测定表明,纯化的Arg比活性为310 IU/mg。结论：成功构建了Arg的毕赤酵母高效表达菌种,建立了目标物质的分离纯化工艺。     

中华鲟半胱氨酸蛋白酶抑制剂在毕赤酵母中的表达和活性分析

为研究鱼类半胱氨酸蛋白酶抑制剂（Cystatin）的功能并探索其在水产加工和病害防治中的应用潜力,将PCR改造后的编码成熟肽中华鲟（Acipenser sinensis）cystatin 基因亚克隆到毕赤酵母整合型表达载体pPICZαA,氯化锂法转化毕赤酵母菌株GS115,构建表达cystatin的酵母基因工程菌。经甲醇诱导、SDSPAGE检测培养基上清液,表明中华鲟cystatin在毕赤酵母中实现了高效表达,重组cystatin表达量约为215mg·L^-1。纯化后重组蛋白纯度达94.2%。生物活性检测结果表明,1μg重组中华鲟cystatin约能抑制15μg木瓜蛋白酶的水解活性。     

纤维连接蛋白C端肝素结合域多肽在毕赤酵母中的表达、纯化及鉴定

为在毕赤酵母中表达纤维连接蛋白C端肝素结合域(Fibronectin C-terminal heparin-binding domainFNCHBD)多肽并研究其功能,通过PCR技术扩增FNCHBD目的基因,将目的基因与T载体连接,经测序正确后,插入pAo815SM酵母表达载体增加基因拷贝数,然后酶切克隆入酵母表达载pPIC9K;将重组质粒Sal I酶切线性化后转化毕赤酵母菌株,筛选工程菌,经甲醇诱导表达,用SDS-PAGE检测发酵上清液,表明有重组蛋白FNCHBD多肽的高表达,表达产物通过离心、超滤、离子交换层析纯化,纯化产物通过SDS-PAGE、Western blotting印迹、质谱及肝素亲和层沉析对表达产物进行鉴定。结果表明利用酵母工程菌成功表达和纯化了FNCHBD多肽,多肽的分子量接近32 kDa,纯化产物的纯度可达95%以上,能被FN多克隆抗体特异识别且具有多肽肝素结合活性,为后续结构及功能的研究奠定基础。     

日本囊对虾Kunitz型蛋白酶抑制剂在毕赤酵母中的表达纯化及活性分析

SKPI(shrimp Kunitz-type protease inhibitor)是日本囊对虾(Marsupenaeus japonicus)体内的一个小分子多肽, 含有一个Kunitz型结构域, 属于丝氨酸蛋白酶抑制剂。目前已知丝氨酸蛋白酶抑制剂在节肢动物免疫系统中起着非常重要的作用, 为了了解SKPI在对虾天然免疫系统中的作用, 首先对其进行了重组表达。从日本囊对虾肝胰腺中扩增skpi的cDNA片段, 插入改造后的pPIC9K酵母表达载体, 获得的重组质粒转化至毕赤酵母GS115进行表达。由于改造的pPIC9K载体加入了6-His标签, 因此利用Ni?Sepharose?High?Performance对SKPI进行了高效纯化。初步的活性研究表明, 重组表达的SKPI能特异性地抑制胰蛋白酶的水解活性。     

日本囊对虾Kunitz型蛋白酶抑制剂在毕赤酵母中的表达纯化及活性分析

SKPI(shrimp Kunitz-type protease inhibitor)是日本囊对虾(Marsupenaeus japonicus)体内的一个小分子多肽, 含有一个Kunitz型结构域, 属于丝氨酸蛋白酶抑制剂。目前已知丝氨酸蛋白酶抑制剂在节肢动物免疫系统中起着非常重要的作用, 为了了解SKPI在对虾天然免疫系统中的作用, 首先对其进行了重组表达。从日本囊对虾肝胰腺中扩增skpi的cDNA片段, 插入改造后的pPIC9K酵母表达载体, 获得的重组质粒转化至毕赤酵母GS115进行表达。由于改造的pPIC9K载体加入了6-His标签, 因此利用Ni?Sepharose?High?Performance对SKPI进行了高效纯化。初步的活性研究表明, 重组表达的SKPI能特异性地抑制胰蛋白酶的水解活性。     

牛凝乳酶基因在毕赤酵母中的重组表达

通过PCR技术从克隆载体pMD18T-Prochy上扩增牛凝乳酶原基因,双酶切后定向插入到酵母表达载体pPICZaA中,构建表达质粒pPICZaA-Prochy,线性化后电转化毕赤酵母GS115,经PCR和测序鉴定凝乳酶原基因成功插入到毕赤酵母的基因组中。在甲醇诱导下进行凝乳酶的表达,SDS-PAGE分析证明重组凝乳酶的分子量约为37 kD,培养基上清液中凝乳酶的活性为12.2 SU/mL。本研究首次应用毕赤酵母表达牛凝乳酶,在培养基中获得分泌表达的重组凝乳酶,为干酪工业提供了新型及优良的凝乳酶来源。     

重组hIL-10质粒构建及其在毕赤酵母SMD1168中的表达和纯化

构建重组hIL-10真核表达载体,探索在毕赤酵母中的表达,为进一步研究hIL-10生物学功能及临床应用奠定基础。PCR扩增目的基因hIL-10cDNA,经EcoRI/XbaI双酶切后,将其亚克隆至同样双酶切的载体pPICZaA中,构建表达质粒pPICZaA-hIL-10;电转化法,将其转入毕赤酵母SMD1168,甲醇诱导Mut+型转化子基因表达;对目的蛋白进行检测及纯化。扩增出了目的基因hIL-10cDNA,重组质粒pPICZaA-hIL-10经酶切鉴定和序列测定正确;表达产物经SDS-PAGE分析在42.0kD处可见较浓染条带,Westernblotting分析可见特异条带;ELISA检测72h上清液中目的蛋白浓度可达1.973mg/L;纯化后目的蛋白纯度达67.0%。实现了重组hIL-10在毕赤酵母SMD1168中的成功表达和初步纯化。     

极细链格孢菌peaT1基因在毕赤酵母中的表达与功能 分析

建立了在毕赤酵母(Pichiapastoris)中分泌表达PeaT1蛋白的技术。将来源于极细链格孢菌(Alternaria tenuissima)的基因peaT1亚克隆至酵母分泌型表达载体pPIC9K,构建了重组表达载体pPIC9K-peaT1,分别用SalI或BglII酶切线性化后电击转入毕赤酵母GS115菌株中,经MD平板筛选、PCR鉴定获得整合有外源基因的重组菌株。在α-Factor及AOX1基因启动子和终止信号的调控下,PeaT1在酵母中大量表达并分泌到胞外,SDS-PAGE检测表明表达蛋白的表观分子量约为35kD。表达蛋白上清稀释液能诱导烟草产生对TMV的抗性,其枯斑数抑制率可达到30.37%。每升表达上清液经超滤浓缩和离子交换层析可纯化目的蛋白16.13mg,该纯化蛋白能显著地促进小麦幼苗的生长。     

定点突变巴曲酶在毕赤酵母中的克隆与表达

目的 为避免毕赤酵母分泌的Kex2蛋白酶对发酵液中所表达的巴曲酶的降解.利用重叠PCR方法对巴曲酶基因进行定点突变,将巴曲酶基因第45位的Arg突变为Lys.方法 将突变后的基因克隆到酵母分泌型表达载体pPICZαA中,将重组载体酶切线性化后经电转化转入巴斯德毕赤酵母细胞,筛选鉴定转化子,经摇瓶发酵甲醇诱导,酵母菌分泌表达有凝血活性的定点突变巴曲酶,经SDS-PAG电泳、免疫印迹确定其分子量为32 kD.结果发酵罐的表达量达到52 KU/ml发酵液,较重组天然巴曲酶的表达量提高了73.3%.结论 定点突变巴曲酶的表达量比重组天然巴曲酶的表达量有显著提高,表达的突变巴曲酶同样具有凝血活性.     

Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens.

Elafin (elastase-specific inhibitor) is a low molecular weight inhibitor of neutrophil elastase which is secreted in the lung. Using synthetic peptides corresponding to full-length elafin (H2N-1AVT.....95Q-OH), the NH2-terminal domain (H2N-1AVT.....50K-OH) and the COOH-terminal domain (H2N-51PGS.....95Q-OH), we demonstrate that elafin's anti-elastase activity resides exclusively in the COOH-terminus. Several characteristics of elafin suggest potential anti-microbial activity. The anti-microbial activity of elafin, and of its two structural domains, was tested against the respiratory pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Elafin killed both bacteria efficiently, with 93% killing of P. aeruginosa by 2.5 microM elafin and 48% killing of S. aureus by 25 microM elafin. For both organisms, full-length elafin was required to optimise bacterial killing. These findings represent the first demonstration of co-existent anti-proteolytic and anti-microbial functions for elafin.     

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.     

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis

Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.     

Elafin is a potent inhibitor of proteinase 3

Elafin, a human skin derived inhibitor of human leukocyte elastase, was tested for inhibitory activity against proteinase 3, an elastin degrading proteinase of neutrophils. The inhibitory activity of elafin was compared with antileukoprotease and eglin C. Elafin proved to be a potent inhibitor of elastin-FITC degradation showing an IC 50 of 9.5 x 10(-9) M. Potency was found to be more than 100-fold higher as compared with antileukoprotease and eglin C.     

Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor

Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.     

Recombinant human elafin protects airway epithelium integrity during inflammation

Elafin, an antiprotease, is likely to protect pulmonary epithelial cells from inflammatory damages. We aimed to explore the molecule mechanisms of recombinant human elafin on protecting A549 cells integrity against inflammatory assault. We transfected A549 airway epithelial cells with eukaryotic expression vector pEGFP-N1-Elafin and negative control vector pEGFP-N1, respectively. Cells were co-incubated with polymorphonuclear neutrophils (PMN) and then were stimulated with lipopolysaccharide (LPS). Results revealed that, in pEGFP-N1-Elafin transfected cells, neutrophil elastase (NE) activity significantly decreased after LPS stimulation, accompanied with elevated elafin mRNA level and protein production, whereas in cells transfected with pEGFP-N1, NE activity was higher and elafin expression was lower, compared with pEGFP-N1-Elafin transfected group (P < 0.05). In pEGFP-N1 transfected cells, LPS suppressed tight junctions protein zonula occludens-1 (ZO-1) production, while in recombinant pEGFP-N1-Elafin cells, LPS did not cause significantly decrease of ZO-1, compared with normal control cells. LPS stimulation also significantly weakened the collagen adhesion capability of pEGFP-N1 transfected cells (P < 0.01), but there was no significant difference in recombinant pEGFP-N1-Elafin cells (P > 0.05). These results suggest that elafin can maintain airway epithelium integrity by protecting airway epithelial cells and enhancing the anti-inflammatory capability of airway.