Construction of a protease-deficient strain set for the fission yeast Schizosaccharomyces pombe, useful for effective production of protease-sensitive heterologous proteins

One of the major problems hindering effective production and purification of heterologous proteins from the fission yeast Schizosaccharomyces pombe is proteolytic degradation of the recombinant gene products by host-specific proteases. As an initial solution to this problem, we constructed a protease-deficient disruptant set by respective disruption of 52 Sz. pombe protease genes. Functional screening of the resultant set was performed by observing secretory production of a proteolytically sensitive model protein, human growth hormone (hGH). The results indicated that some of the resultant disruptants were effective in reducing hGH degradation, as observed during the hGH expression procedure and mainly as a result of unknown serine- and/or cysteine-type proteases in the culture medium. These findings also demonstrated that construction of a protease-deficient strain set is not only useful for practical application in protein production, but also for functional screening, specification and modification of proteases in Sz. pombe, where further investigations of proteolytic processes and improvement through multiple gene manipulations are required.     

Efficient production of an antibody Fab fragment using the baculovirus–insect cell system

The production of an Fab fragment of the catalytic antibody 6D9 in lepidopteran insect cells infected with a recombinant baculovirus that contained both the heavy chain (Hc) and light chain (Lc) genes of the Fab fragment was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) of culture supernatant showed that baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted an Fab fragment that retained antigen-binding activity. Infection of High Five cells with a recombinant baculovirus, in which the Lc and Hc genes were located downstream of the baculovirus p10 and polyhedrin promoters, respectively, produced a higher Fab fragment yield than that obtained with a baculovirus in which the Hc and Lc genes were downstream of the p10 and polyhedrin promoters, respectively. Baculovirus-infected High Five cells secreted more of the Fab fragment than Spodoptera frugiperda Sf9 cells. Moreover, use of the baculovirus gp64 signal sequence upstream of the Lc and Hc genes resulted in greater yield of the secreted Fab fragment than use of the insect-derived BiP and melittin signal sequences. Consequently, the Fab fragment was obtained in a high yield (> 600 μg/ml) in a shake-flask culture of High Five cells infected at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell with the recombinant baculovirus in which the Lc and Hc genes with the gp64 signal sequence were expressed under the control of the p10 and polyhedrin promoters, respectively. These results indicate that the baculovirus–insect cell system may allow efficient production of antibody Fab fragments.     

不同蛋白酶突变子对鲢鱼蛋白酶解及产物抗氧化性的影响

本实验采用芽孢杆菌丝氨酸蛋白酶及其突变产物对淡水鱼类白鲢鱼肉蛋白进行酶解,对比分析了酶的不同突变产物对水解过程水解度的影响及其酶解产物的抗氧化活性变化。大部分蛋白酶在水解反应前40 min内反应速率较高,水解度迅速增加,40min~120 min之间水解曲线变得平缓甚至下降。其中细菌丝氨酸蛋白酶突变子239对白鲢鱼肉蛋白的水解能力最强,其2 h水解产物水解度最大,为11.51%;突变蛋白酶33的水解能力最差,最终水解产物的水解度只有3.08%。细菌丝氨酸蛋白酶及其突变酶的酶解产物均具有较高的抗氧化性,其中蛋白酶突变子166的酶解产物抗氧化活性最强,经测定其DPPH清除能力为91.02%,蛋白酶突变子169的酶解产物抗氧化活性最弱,DPPH清除能力为61.41%。结果表明,与三联体催化活性中心相关的突变对丝氨酸蛋白酶的水解能力和水产品蛋白的水解度影响较大,而酶底物特异性方面的改变则影响酶解产物的抗氧化活性。     

抗牛免疫球蛋白G单克隆抗体的制备及鉴定

采用杂交瘤技术从90株融合株中筛选得到分泌抗牛免疫球蛋白G（immunoglobulin G,IgG）单克隆抗体的2F6、5E3、9C6、9H8四个细胞株。其抗体亚型均为IgG1型,腹水单克隆抗体的效价可达5.12×106;5E3、9H8识别牛IgG重链Fc片段,2F6识别牛IgG轻链Fab片段,9C6识别牛IgG重链Fab片段;9C6与牛IgG1和牛IgG2反应,与山羊IgG、绵羊IgG、兔IgG、牛IgM、牛乳酪蛋白、牛乳β-乳球蛋白、牛乳铁蛋白、牛血清白蛋白、鸡卵清白蛋白、鱼明胶的交叉反应率均小于1%;9C6亲合力常数达牛乳8.92×108?L/mol。     

鱿鱼肝脏蛋白酶的鉴定及组织蛋白酶L的分离纯化

鱿鱼肝脏含有丰富的蛋白酶,为利用其内源蛋白酶进行可控的酶解,本研究以鲤鱼肌原纤维蛋白为底物对鱿鱼肝脏内源蛋白酶的种类和性质进行了研究。反应体系中添加E-64、1,10-菲啰啉和苯甲基磺酰氟(phenylmethylsulfonyl?fluoride,PMSF)后,肌球蛋白重链(myosin?heavy?chain,MHC)的降解得到了显著抑制,确定了鱿鱼肝脏含有金属类、半胱氨酸类、丝氨酸类3类蛋白酶。半胱氨酸类蛋白酶热稳定性最好,在50℃以上仍然具有较大活性,可将肌原纤维蛋白酶解成小分子质量的降解产物。利用特异性底物对半胱氨酸蛋白酶种类进行鉴定发现,该酶只酶解Z-Phe-Arg-MCA,添加亮抑酶肽后相对酶活性为0%,添加E-64相对酶活性仅存0.6%,初步确定鱿鱼肝脏中的半胱氨酸蛋白酶主要为组织蛋白酶L。最后,通过硫酸铵沉淀、离子交换层析、凝胶过滤对组织蛋白酶L进行分离纯化,在电泳上得到了分子质量约为25?kD单一条带。     

Stable earthworm serine proteases: application of the protease function and usefulness of the earthworm autolysate

The fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N. et al., Biosci. Biotechnol. Biochem., 57, 1726-1730 (1993), 60, 293-300 (1996), and 63, 2031-2033 (1999)] were further characterized to exploit their catalytic functions. These enzymes are stable in solution for long periods at room temperature and strongly resistant to organic solvents, even toluene and n-hexane. The serine proteases can act on various protein substrates such as elastin and hemoglobin as well as fibrin, and also catalyzed the hydrolysis of esters such as ethyl acetate and a bioplastic, poly[(R)-3-hydroxybutyrate] film. The enzymes, in the absence of microbial degradation, contributed to the production of the earthworm autolysate possessing antioxidant ability and protease activity, whose components were similar to those of soy sauce. The extract of the earthworm autolysate could be used as a peptone substitute in media for the cultivation of microorganisms.     

Production of Fab fragment corresponding to surface protein antigen of Streptococcus mutans serotype c-derived peptide by Escherichia coli and cultured tobacco cells

The cDNA of a mouse Fab fragment was cloned from a hybridoma cell line that produces a mouse monoclonal antibody, KH5, that reacts with the peptide fragment of the surface protein antigen of Streptococcus mutans serotype c (PAc). After transfection with cDNA, recombinant Fab fragments were produced by Escherichia coli (T15 Fab) and cultured tobacco cells (X253 and X262 Fabs). The antipeptide activities of T15 and X253 were similar to that of KH5. X253 was secreted into the culture media, which had a specific affinity for the PAc peptide.     

HYDROLYSIS OF SALMON MUSCLE PROTEINS BY AN ENZYME MIXTURE EXTRACTED FROM ATLANTIC SALMON (SALMO SALAR) PYLORIC CAECA

An extract of serine proteases from thepyloric caeca of Atlantic salmon (Salmo salar) was recovered by (NH₄)₂SO₄ and stabilized in 20% gfycerol. Proteolytic activity was determined using Azocoll and hydrofytic efficiency evaluated on salmon muscle mince. Activity assays identified chymotrypsin as the most active serine protease component, followed by trypsin and elastase. The caeca extract more efficiently hydrolyzed a salmon muscle mince substrate at 40C compared to 21.5C, and was very efficient compared to four commercial protease preparations in terms of degrees of hydrolysis (DH) achieved after a 180 min reaction. Salmon protein hydrotysates made from the enzymatic hydrolysis with the pyloric caeca enzymes had different peptide profiles at the same DH when the reaction took place at 2I.5C compared to 40C, suggesting that relative activities of these serine proteases may be different at different reaction temperatures.     

Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe

Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic strains of Streptococcus. Expression of streptokinase has been so far attempted in Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that was either highly glycosylated or degraded. Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we decided to express the streptokinase gene in this yeast. A chimeric gene comprising the signal sequence of the Plus pheromone of Sz. pombe fused in-frame with the mature streptokinase from Streptococcus sp. was constructed and inserted into the expression vector containing the thiamine-regulated promoter. We obtained a high level of expression of streptokinase comparable to that in E. coli and P. pastoris, with 50-100% processing of the signal sequence and secretion of the mature streptokinase into the periplasmic fraction. The mature enzyme co-migrates with the authentic mature SK in SDS gels, lacks any major modification and is functional. Importantly, a higher level of expression under stationary phase conditions and improved extractability of the mature and undegraded streptokinase was achieved in a novel mutant of Sz. pombe defective for a potent extracellular protease activity. We suggest that the unique vector/strain system developed here could be advantageous for large-scale production of prokaryotic proteins without significant modification or degradation in Sz. pombe.     

产细菌素植物乳杆菌菌株的筛选及其细菌素生物学特征研究

从甘蓝泡菜中分离到６９株乳酸杆菌,通过琼脂点扩散交叉拮抗试验,筛选出３株有明显抑菌活性代谢产物的植物乳杆菌。排除酸、过氧化氢等干扰因素后,离心发酵液对指示菌Ｌａｃｔｏｂａｃｉｌｕｓｐｌａｎｔａｒｕｍ９６Ｄ仍有抑菌作用,用胰蛋白酶对其透析液处理后活性丧失,说明它们产生的抑菌物质是细菌素。以Ｇ８菌株为试材,对其细菌素类物质的产生及细菌素粗提物进一步研究,发现在对数末期其抑菌活性最高,对热相对稳定（１００℃,２０ｍｉｎ）,易被胰蛋白酶、蛋白酶Ｋ和胃蛋白酶失活,显示活性的ｐＨ值范围为４．０～５．５,粗提液表现为不仅抗明串株菌属、片球菌属、乳杆菌属的一些菌株,而且抗一些非乳酸菌的革兰氏阳性菌,但对大肠杆菌（Ｅ．ｃｏｌｉ）等革兰氏阴性菌没有任何抑制作用。说明Ｇ８菌株产生的是一类抗广谱革兰氏阳性菌的细菌素     

凝胶过滤层析一步纯化MC-LR Fab的方法及吸附机制初探

为高效制备高纯微囊藻毒素(MC-LR)Fab片段,采用木瓜蛋白酶酶解腹水中纯化获得的单克隆抗体,利用该Fab片段与凝胶过滤层析介质结合的特殊现象分离酶解混合物。通过单因素实验考察流动相中离子强度和pH对Fab片段在柱中保留行为的影响,结合Fab片段等电点(pI)及聚集性质分析二者结合机制。结果表明,腹水经protein G纯化得到纯度大于90%的IgG,IgG经木瓜蛋白酶酶解,主要产物为Fab片段、Fc片段和少量IgG,酶解混合物经凝胶过滤层析一步纯化获得纯度为94.5%,回收率为51%,对MC-LR抑制率为94%的Fab片段。Fab片段pI为6.02,由于表面较强疏水性产生聚集,疏水吸附导致Fab片段与凝胶过滤层析介质结合,降低流动相中离子强度,减弱疏水作用同时提高pH增强离子排阻作用可使Fab片段消除吸附,实现了Fab片段的高效制备。     

Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.     

Screening of strains of Lactococci isolated from Egyptian dairy products for their proteolytic activity

A collection of cocci isolates (293) obtained from traditional Egyptian dairy products collected from four Egyptian regions yielded 151 lactic acid bacteria (LAB) cocci isolates. Among them, 24 isolates were characterised as highly proteolytic. SDS–PAGE showed that 6 isolates were the most proteolytically active, which were classified into Enterococcus faecalis HH22 (4 isolates) and Enterococcus faecium DO623 (2 isolates). The proteolytic activity of E. faecalis was higher than that of E. faecium (particularly on β-casein). The maximal degradation of milk proteins was achieved at pH 6.5–7.2 (E. faecalis) or pH 6.5 (E. faecium) and at 42 °C for both strains. The proteolytic activities of the two strains were inhibited mostly by the presence of EDTA, showing that their proteases belong mainly to metalloproteases. A slight inhibition of proteolysis by PMSF in the case of E. faecalis HH22 suggests a limited inclusion of serine proteases in its protease system.     

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu2+ into mesoporphyrin

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.     

Characterisation of a milk-clotting extract from Balanites aegyptiaca fruit pulp

Milk-clotting proteases were extracted and characterised from Balanites aegyptiaca fruit pulp traditionally used in sub-Saharan countries to thicken gruel. The aim was to improve the extraction conditions and milk-clotting in the context of future cheese making. B. aegyptiaca fruits were disinfected, husked and soaked in different buffers and saline solutions. The extracts obtained were diafiltered, concentrated and decoloured with active charcoal. The effects of extract concentration, milk pH and temperature on clotting-time were studied. The use of specific inhibitors showed that the B. aegyptiaca extract contains two types of proteases: an aspartic protease and a serine protease, with an optimum activity at pH 5.0 and pH 8.0, respectively, and an optimal temperature at 50 °C. The proteolytic activity is more thermostable than bovine chymosin at pH 5.0. Partial purification by anion exchange chromatography and sodium docecylsulphate polyacrylamide gel electrophoresis allowed separation of the two milk-clotting proteases.     

Characterisation of proteolytic enzymes from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii)

BACKGROUND: Fresh water prawn in Thailand is widely consumed due to its delicacy. During postmortem handling and storage, prawn meat becomes soft and mushy, probably as a result of indigenous proteases. Therefore, an understanding of prawn proteases associated with the degradation of muscle proteins from fresh water prawn could pave the way for prevention of such a phenomenon during extended storage. RESULTS: Proteolytic enzymes in the crude extract (CE) from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) were characterised. CE from muscle exhibited the highest hydrolytic activities towards haemoglobin at pH 5 and 50 °C, while that from hepatopancreas had the highest activity on casein at pH 7 and 60 °C. Based on inhibitor study, cysteine protease and serine protease were dominant in CE from muscle and hepatopancreas, respectively. CE from muscle rarely hydrolysed natural actomyosin (NAM), but could not degrade pepsin‐soluble collagen (PSC). Conversely, NAM and PSC were susceptible to hydrolysis by CE from hepatopancreas as evidenced by the marked decreases in band intensity. Activity staining using haemoglobin, casein and gelatin as substrates revealed that no proteolytic or gelatinolytic activity was observed in CE from prawn muscle, while CE from hepatopancreas exhibited pronounced hydrolytic activities towards all substrates. CE from muscle showed calpain and cathepsin L activities but CE from hepatopancreas mainly exhibited tryptic and chymotryptic activities. CONCLUSION: Serine proteases, mainly trypsin‐like or chymotrypsin‐like, from hepatopancreas were probably responsible for the softening of prawn meat during postmortem storage via the degradation of both muscle and connective tissues. Copyright © 2010 Society of Chemical Industry     

Bacillus subtilis AprX involved in degradation of a heterologous protein during the late stationary growth phase

In Bacillus subtilis, extracellular protease-deficient mutants have been used in attempts to increase the productivity of heterologous proteins. We detected protease activity of AprX using protease zymography in the culture medium at the late stationary growth phase. An alpha-amylase-A522-PreS2 hybrid protein, in which the PreS2 antigen of human hepatitis B virus (HBV) is fused with the N-terminal 522-amino-acid polypeptide of B. subtilis alpha-amylase, has been produced in multiple-protease-deficient mutants. The B. subtilis KA8AX strain, which is deficient in eight extracellular proteases and AprX, did not show the proteolysis of alpha-amylase-A522-PreS2 in the late stationary growth phase. Moreover, the production of alpha-amylase-A522-PreS2 was about 80 mg/l, which was eight times higher than that by the KA8AX strain previously reported. In addition, we showed the degradation of the heterologous protein by AprX that leaked to the culture medium (probably caused by cell lysis) during the late stationary growth phase.     

刺参2 种天冬氨酸蛋白酶的酶学性质及其对自溶的影响

对刺参体内2?种天冬氨酸蛋白酶——组织蛋白酶D（cathepsin?D,Cat?D）和组织蛋白酶E（Cat?E）的酶学性质进行探讨,并考察两者可能在刺参自溶中的参与作用。先用Tris-HCl缓冲溶液提取刺参体壁中的粗酶,采用特异性荧光底物法测定Cat?D和Cat?E的酶学性质。结果表明,Cat?D和Cat?E的最适pH值分别为5.0和4.0,分别在60?℃和40?℃呈现最大酶活性,两者在20～40?℃活性均较为稳定。Zn2＋、Cu2＋、Fe2＋、Fe3＋、Mn2＋可抑制Cat?D的活性,抑制率分别为86%、76.3%、29.2%、56.5%和48.5%。Fe3＋、Fe2＋、Cu2＋可抑制Cat?E的活力,抑制率分别为99.1%、82.2%和28.6%。Pepstatin?A、Z-Leu-Leu-Leu-H、苯甲基磺酸氟、1,10-菲啰啉能够抑制两者活性,抑制率分别约为98%～99%、65%～78%、30%～35%、19%～23%。二硫苏糖醇、L-Cys和乙二胺四乙酸则可将两者活性分别提升30.4%～31.1%、7.58%～9.64%、6.6%～7.9%。结果表明,刺参Cat?D和Cat?E为2?种具有一定金属离子敏感性和依赖性的天冬氨酸蛋白酶,其活性中心有丝氨酸和半胱氨酸参与其活性调节,且两者很有可能参与刺参自溶过程中蛋白质的降解。