短芽孢杆菌细胞壁蛋白基因多启动子和信号肽编码序列的分离

具有高蛋白分泌能力的短芽孢杆菌分泌到胞外的蛋白质主要是细胞壁蛋白,本通过PCR从5株筛得的具有高蛋白分泌能力且没有胞外蛋白酶活性的短芽孢杆菌中分离出细胞壁蛋白基因多启动子和信号肽编码序列,对其分析发现与具有高蛋白分泌能力的短芽孢杆菌47和HPD31的相应序列高度同源,该结果表明分泌蛋白能力强的短芽孢杆菌细胞壁蛋白的合在 可能受同样的机理调控。     

滇重楼寄生菌的研究

从滇重楼(Paris polyphylla var.yunnanensis)地下茎中分离和鉴定出两种细菌——蜡状芽孢杆菌(Bacillus cereus)和产碱假单胞菌(Pseudomonas alcaligenes),以及三种真菌——黑团孢霉(Periconia sp.)、白色厚顶孢霉(Pachnocybe albida)和重楼索霉(Hormomyces paridiphilus)。对蜡状芽孢杆菌、产碱假单胞菌和重楼索霉进行了液体培养并测定了胞外多糖含量,结果表明重楼索霉可分泌大量胞外多糖,这可能是导致滇重楼地下茎胶质化和多糖含量增加的原因。     

丝状真菌表达分泌系统中受体菌的构建

黑曲霉糖化酶高产菌株T21经紫外诱变后, 通过酪蛋白平板和蛋白酶活性测定筛选出胞外酸性蛋白酶活力仅为原株076%的菌株A.nigerT21-201,其生长特性和产糖化酶活力与原株基本一致。利用原生质体PEG法将含有报告基因vhb的表达分泌质粒Pgt10-vhb通过与选择标记质粒的共转化导入此蛋白酶部分缺陷株及其原株T21,检测在蛋白酶缺陷株Aspergillus niger T21-201 和原株T21中VHb的分泌表达,结果表明在A.nigerT21-201中VHb表达水平显著高于原株,但Northern blot却显示在两菌株中^vnb基因的转录水平近似,由此证明酸性蛋白酶缺陷对保护外源蛋白产生了显著效果。      

短短小芽孢杆菌大肠杆菌穿梭分泌表达载体的构建

应用PCR技术从具有分泌蛋白能力强且没有胞外蛋白酶活性的短短小芽孢杆菌50中分离出细胞壁蛋白基因的多启动子和信号肽编码序列,利用它与质粒pUB110和pKF3-起构建成穿梭分泌表达载体pBKE50,将α0淀粉酶基因引入该载体转化短短小芽孢杆菌50后,发现α－淀粉酶可以活性形式分泌表达,此工作为下一步建立短短小芽孢杆菌高效分泌表达系统奠定了基础。     

黑曲霉蛋白酶缺陷突变体的分离和鉴定

对黑曲霉(Aspergillus niger)3.795进行紫外诱变和筛选,获得了胞外蛋白酶缺失的菌株。其中突变株37951、3795123和3795130的生长特性与原株基本相同,胞外蛋白酶的活性明显低于原株,葡萄糖淀粉酶的活性与原株基本相同。突变株3795123和3795130的胞外蛋白酶活性分别为原株的54%和84%,可作为外源基因高效分泌表达的宿主菌。     

多肽抗生素apidaecin基因在乳酸乳球菌中的融合表达

利用乳链菌肽(nisin)诱导表达系统,以泛素(ubiquitin)融合蛋白的形式在乳酸乳球菌(Lactococcus lactis)中表达了多肽抗生素apidaecin。利用TricineSDSPAGE和Western blotting均可在诱导后的宿主菌中检测到特异蛋白带。表达产物的最高产量可达宿主菌可溶性蛋白的7.2%左右。在体外用泛素特异性蛋白酶UBPI从融合蛋白中切除泛素后,产物具有明显的抗菌活性。     

一株地衣芽孢杆菌碱性蛋白酶的研究I.

从皖北盐碱地土样中分离到 011菌株 ,对其进行了菌种鉴定以及胞外碱性蛋白酶的初步研究。结果表明 :011菌株符合地衣芽孢杆菌种的特征 ,但该菌芽孢端生、孢囊膨大、中度耐盐、高度耐碱 ,可在NaCl浓度为 13%和pH11的培养基中生长 ,这些特征又不同于该种几个模式株 ,因而将 011菌株鉴定为地衣芽孢杆菌的一个亚种 ,命名为地衣芽孢杆菌砀山亚种(Bacilluslicheniformissubsp .dangshanensis)。该菌在发酵培养基中能产生较高产量的胞外碱性蛋白酶 ( 72     

一株拮抗多重耐药菌的芽孢杆菌的初步研究

目的 对一株具有拮抗多重耐药菌的芽孢杆菌的特性进行研究.方法 采用菌落法测试多株芽孢杆菌对18种不同来源致病菌的抑制效应,并对其中一株可拮抗多重耐药菌的芽孢杆菌(KC株)进行鉴定和电泳分析.结果 该芽孢杆菌(KC株)经生理生化以及16S rDNA鉴定为枯草芽孢杆菌;KC株对人源、鱼源、畜禽源和鸡粪源的多重耐药菌有不同程度的拮抗效应;SDS-PAGE分析提示,35 kD左右的分泌蛋白可能参与了KC株的抑菌活性.结论 具有拮抗多重耐药菌的芽孢杆菌为枯草芽孢杆菌,且该菌分泌的35 kD蛋白可能参与抑菌活性.     

芽胞杆菌中重组蛋白分泌表达的优化策略及研究进展

芽胞杆菌属具有良好的蛋白表达和分泌能力,在工业酶的生产中被广泛应用,是理想的工业宿主菌,但实现蛋白分泌表达的普遍高效性还存在许多瓶颈。本文综述了芽胞杆菌的蛋白分泌表达策略,从启动子、信号肽、分泌途径、宿主和培养条件这5个方面总结了提高芽胞杆菌中分泌表达重组蛋白的方法,对芽胞杆菌高效生产工业酶有一定的参考价值,最后展望了优化芽胞杆菌分泌表达的研究方向,各种新型生物技术的发展必将推进芽胞杆菌在分泌表达领域有更深入的应用。     

外源蛋白在芽孢杆菌中分泌表达的研究进展

芽孢杆菌是很有潜力的分泌型基因工程宿主菌。本文概述了利用芽孢杆菌分泌表达外源基因时,影响目的蛋白产率的一些主要因素,如蛋白酶水解作用、缺乏适宜的分子伴侣、信号肽的选择不当等,并讨论了相应的解决对策。     

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.     

Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies

Cellular lysis properties of strains of Bacillus subtilis deficient in the synthesis of extracellular proteases was investigated. In all cases, extracellular protease deficiency was found to increase the extent of cellular lysis of batch cultured strains following the transition to stationary phase, the time at which extracellular degradative enzymes are secreted in large quantities. The data indicates that the major extracellular proteases, NprE and AprE, are primarily responsible for the control of this autolytic activity in B. subtilis and has implications for the use of extracellular protease-deficient strains as hosts for the production of heterologous proteins.     

Plasminogen activator: The major secreted neutral protease of cultured skeletal muscle cells

Clonal mouse skeletal muscle cells which differentiate in culture and from synpases with neuronal cells were found to secrete high levels of protease activity as measured with an ¹²⁵I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean typsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occured with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromusclar contacts in experimental and pathologic denervation.     

A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins.     

Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris

The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis‐sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Δvps8 and Δvps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography‐mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Δvps8 and Δvps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Δvps21Δprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.     

The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.     

Production of active Serratia marcescens metalloprotease from Escherichia coli by alpha-hemolysin HlyB and HlyD.

Serratia marcescens produces an abundant extracellular metalloprotease. The gene for this protease had previously been cloned and expressed in Escherichia coli, in which no functional protease could be found. However, the protease gene carries the LXGGXGND repeat motif found in alpha-hemolysin and other proteins secreted by homologous systems. Using a dual-plasmid complementation system, we show that the alpha-hemolysin hlyB and hlyD transport determinants are sufficient to allow secretion and activation of a functional metalloprotease species from E. coli, as are the comparable protease secretion functions of Erwinia chrysanthemi. However, strains expressing protease with the hlyBD transport system are unstable and rapidly lose the ability to produce functional protease.     

Higher production of rabies virus in serum-free medium cell cultures on microcarriers

The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA1 strain of B. thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein. No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis.     

A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.