重组羧肽酶原B的复性方法研究

构建的羧肽酶原B表达质粒在大肠杆菌中获得高表达。但目的蛋白是以包涵体的形式存在。为了获得活性羧肽酶B,必须对其包涵体进行变复性。首先利用稀释复性确定了羧肽酶原B复性的最佳缓冲液;在凝胶过滤复性中,研究了柱长和洗脱流速对羧肽酶原B复性效率的影响;另外对比了稀释复性、透析复性、凝胶层析复性和Ni2 亲合层析法等四种方法对羧肽酶原B的复性效果。结果发现,这4种方法的复性效果有以下顺序:凝胶过滤复性>稀释复性>Ni2 亲合层析>透析复性。     

鼠羧肽酶原B在毕赤酵母中的表达、纯化与鉴定

为了在毕赤酵母中表达鼠羧肽酶原B(procarboxypeptidaseB,proCPB)蛋白,以RT-PCR法从SD鼠胰腺细胞中克隆了proCPB基因,将其插入pPIC9载体,PEG1000介导转入毕赤酵母GS115细胞,在甲醇的诱导下,实现了proCPB在毕赤酵母中的成功表达。通过发酵条件的优化,使用BMGY(pH6·0)培养基,添加0·5%的酪蛋白水解物,于28℃,在起始OD600达10·0时,每隔12h补加0·5%的甲醇,重组酵母GS115-proCPB表达的产物量可达到最高(500mg/L),表达时间可达120h,表达的目的蛋白占总蛋白的94%以上。通过纯化条件的优化,采用两步疏水层析,可使目的蛋白的纯度达96%以上,蛋白得率达38%,重组proCPB活化后所得CPB的比活力可达110u/mg(CPB标准品为180u/mg)。相对分子量测定表明重组蛋白的分子量与理论值极相近,N-端氨基酸测序进一步表明proCPB基因在毕赤酵母中得到了正确的表达和翻译后加工修饰。     

重组羧肽酶原B的复性方法研究*

构建的羧肽酶原B表达质粒在大肠杆菌中获得高表达。但目的蛋白是以包涵体的形式存在。为了获得活性羧肽酶B,必须对其包涵体进行变复性。首先利用稀释复性确定了羧肽酶原B复性的最佳缓冲液;在凝胶过滤复性中,研究了柱长和洗脱流速对羧肽酶原B复性效率的影响;另外对比了稀释复性、透析复性、凝胶层析复性和Ni²⁺亲合层析法等四种方法对羧肽酶原B的复性效果。结果发现,这4种方法的复性效果有以下顺序:凝胶过滤复性>稀释复性>Ni²⁺亲合层析>透析复性。     

体外模拟猪胃条件下木聚糖酶活力稳定性研究

木聚糖酶是饲用复合酶制剂的主要酶系之一,本实验对某木聚糖酶在体外模拟猪胃条件下酶活力的稳定性进行了研究.结果表明:实验所用酶最适温度为50℃左右,最适pH为5.5左右;酶的耐热性能比较好,pH稳定性范围为5～8,体外模拟猪胃条件下木聚糖酶稳定性实验表明,酶活损失较大,经不同方法处理木聚糖酶,酶活损失百分率大小顺序为:胃蛋白酶<新鲜猪胃液<胃蛋白酶+金属离子.     

羧肽酶A1全酶及其活性中心基因的克隆及原核表达

通过RT-PCR方法扩增出CPA1全酶及活性中心基因,分别克隆入载体pGEM-T easy,测序分析。将两段目的基因亚克隆于表达载体pGEX—IT-1,测序证实序列插入正确后转化大肠杆菌BL21,经IPTG诱导表达重组蛋白。结果表明,成功克隆了人CPA1全酶及其活性中心基因并得到了原核表达产物,为进一步分析比较CPA1全酶及其活性中心的特性、了解CPA1结构与功能的关系并最终得到CPA1最小活性结构域奠定基础。     

羧肽酶Y标记蛋白质羧基端方法的优化及应用

蛋白质水解是一种重要的翻译后修饰,它在许多生化过程 (如细胞凋亡和肿瘤细胞转移等) 中起着极其重要的作用。鉴定蛋白质水解位点可以进一步加深我们对这些生化过程的认识。尽管蛋白质氨基端标记方法和蛋白质组学在复杂生物体系中鉴定获得了许多蛋白质的水解位点,但这种方法存在固有的缺陷。羧基端标记方法是另一种可行的鉴定蛋白质水解位点的方法。本文优化了蛋白质羧基端生物酶标记方法,提高了亲和标记效率,从而可以更好地利用正向分离方法对蛋白质羧基端多肽进行分离并用质谱鉴定。我们用优化后的羧基端标记方法来标记大肠杆菌Escherichia coli复杂蛋白样品后鉴定到了120多个蛋白质羧基端多肽和内切多肽。在其所鉴定的蛋白质水解位点中,我们发现了许多已知和未知的位点,这些新的水解位点有可能在正常生化过程的调控发挥着重要的作用。该研究提供了一个可以与蛋白质氨基端组学互为补充、可在复杂体系中鉴定蛋白质水解的方法。     

家蚕蜕皮液羧肽酶A的表征及免疫荧光定位分析

蜕皮是许多变态发育昆虫的一种重要生理现象,昆虫通过蜕皮液中的酶对新旧表皮进行分离。已有相关蛋白组学的研究证明,家蚕蜕皮液中具有一种含量丰富的羧肽酶A(Bombyx mori-carboxypeptidase A, Bm-CPA),目前对其作用功能尚不清楚。为了更好地了解Bm-CPA在家蚕蜕皮发育过程的作用,本研究通过生物信息学分析、实时荧光定量PCR、抗体制备、免疫荧光染色和毕赤酵母表达等方法对Bm-CPA进行了研究。结果显示,Bm-CPA具有保守的M14锌羧肽酶结构域和糖基化位点,并且受蜕皮激素(20-hydroxyecdysone, 20E)调控,在眠期和上簇期的表皮中大量表达;免疫荧光染色显示Bm-CPA在眠期的表皮中富集,Bm-CPA抑制剂会导致幼虫因无法蜕皮而死亡;通过毕赤酵母表达系统在体外成功获得大量的重组Bm-CPA蛋白。这些结果为深入了解家蚕蜕皮发育过程提供了一定的参考。     

通过定点突变提高乳链菌肽对热及pH的稳定性

【目的】通过定点突变技术改变乳链菌肽(nisin)特定位置氨基酸,获得性质改善的nisin突变体,为扩大其应用范围提供依据。【方法】在抑菌谱扩大的nisin单突变体M21K nisinZ的基础上,对M21K nisZ基因第29位丝氨酸密码子进行定点突变;将其克隆至乳酸菌表达载体pMG36e,并在Lactococcus lactis NZ9800中进行表达;双突变体M21K/S29K nisinZ经分离纯化后检测其在抑菌活性、抑菌谱和稳定性等方面的变化。【结果】与单突变体M21K nisinZ及野生型nisinZ(wild-type,WT)相比,双突变体M21K/S29K nisinZ对指示菌的抑菌活性虽有所下降,但其对温度及pH值的稳定性有显著提高。同时其抑菌谱与M21K nisinZ相同,可抑制革兰氏阴性菌,扩大了WT的抑菌谱。【结论】通过改变nisin分子特定位置的氨基酸可以改善nisin分子的理化性质,有可能得到应用范围更广的nisin品种。     

草地螟羧肽酶A基因的克隆、表达及序列分析

羧肽酶是昆虫体内重要的消化酶系之一。利用甜菜夜蛾Spodoptera exigua（Hübner）围食膜多克隆抗体免疫筛选草地螟Loxostege sticticalis L.中肠cDNA表达文库, 得到编码羧肽酶A的全长cDNA克隆。该cDNA克隆全长1 380 bp （GenBank登录号EU924506）, 开放阅读框长1 302 bp, 编码434个氨基酸, 预测分子量和等电点分别为49.1 kDa和9.56。序列含有胰蛋白酶切割位点和催化特征, 具有典型的羧肽酶A特性。将该基因与pET30载体重组后, 经IPTG诱导, 蛋白在大肠杆菌中获得了表达。     

产羧肽酶菌株的筛选及其产酶特性的研究*

从土壤中分离到28株产蛋白酶菌株,用其粗酶液水解多肽并经氨基酸自动分析仪测定产物中游离氨基酸的含量和种类,从中筛选到一株产羧肽酶的菌株。对该菌株的菌落和菌体形态进行观察,确定其属于曲霉菌属。该菌株在发酵至第48h时,发酵液中羧肽酶活性达到最大。此时发酵液pH值为7.46,菌体已处于衰败期,说明该酶的合成方式为延迟合成型。     

Catalytic activity of carboxypeptidase B and of carboxypeptidase Y with anisylazoformyl substrates

Anisylazoformyllysine (CH3OC6H4-N = N-CO-Lys-OH) is rapidly hydrolyzed at the acyl-lysine linkage by the zinc-enzyme porcine carboxypeptidase B. The catalytic reaction is readily monitored spectrophotometrically by disappearance of the intense absorption (348.5 nm, epsilon 18400) of the azo chromophore, which chemically fragments after substrate cleavage. Carboxypeptidase Y has no activity toward this type of substrate.     

Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity

An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening.     

On the pH‐optimum of activity and stability of proteins

Biological macromolecules evolved to perform their function in specific cellular environment (subcellular compartments or tissues); therefore, they should be adapted to the biophysical characteristics of the corresponding environment, one of them being the characteristic pH. Many macromolecular properties are pH dependent, such as activity and stability. However, only activity is biologically important, while stability may not be crucial for the corresponding reaction. Here, we show that the pH‐optimum of activity (the pH of maximal activity) is correlated with the pH‐optimum of stability (the pH of maximal stability) on a set of 310 proteins with available experimental data. We speculate that such a correlation is needed to allow the corresponding macromolecules to tolerate small pH fluctuations that are inevitable with cellular function. Our findings rationalize the efforts of correlating the pH of maximal stability and the characteristic pH of subcellular compartments, as only pH of activity is subject of evolutionary pressure. In addition, our analysis confirmed the previous observation that pH‐optimum of activity and stability are not correlated with the isoelectric point, pI, or with the optimal temperature. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B

The action of bovine spleen cathepsin B as a dipeptidyl carboxypeptidase on newly synthesized substrates of the type peptidyl-X-p-nitrophenylalanyl (Phe(NO2))-Y (X,Y = amino acid residue) or 5-dimethylaminonaphthalene-1-sulfonyl (Dns)-peptidyl-X-Phe(NO2)-Y was investigated. The kinetic parameters of hydrolysis of the X-Phe(NO2) bond were determined by difference spectrophotometry (delta epsilon 310 = 1600 M-1 cm-1) or by spectrofluorometry by following the five- to eightfold increase of Dns-group fluorescence with excitation at 350 nm and emission at 535 nm. The substrates were moderately sensitive to cathepsin B; kcat varied from 0.7 to 4 s-1 at pH 5 and 25 degrees C; Km varied from 6 to 240 microM. The very acidic optima of pH 4-5 are characteristic for dipeptidyl carboxypeptidase activity of cathepsin B. Bovine spleen cathepsins S and H had little and no activity, respectively, when assayed with Pro-Glu-Ala-Phe(NO2)-Gly. These peptides should be a valuable tool for routine assays and for mechanistic studies on cathepsin B.     

Zinc deficiency and dipeptidyl carboxypeptidase activity

Dipeptidyl carboxypeptidase (DC) is highly active in the testis and epididymis of rats and increases during pubertal development. Zinc deficiency during this period depresses the activity of DC in the testis. Experiments were conducted to determine the effects of zinc deficiency on epididymal DC activity. Comparisons were made between changes seen in this organ and those observed in testis. Three dietary treatments were used; zinc-deficient, fed ad libitum; zinc-adequate, pair-fed to the deficient group; and zinc-adequate, fed ad libitum. Results confirmed that testicular DC is affected negatively by zinc deficiency. DC activity was also lower in the epididymis of zinc-deficient rats than in control rats. These effects apparently were specific relative to changes in activity of other enzymes. Alkaline phosphatase activity in the epididymis was not affected by zinc deficiency and it was depressed in the testis. Gamma-glytamyl transferase activity in the epididymis was not affected by zinc deficiency but it was elevated in the testis. The results of this study suggest that part of the effect of zinc deficiency on sexual maturity in the male rat may be caused by reduced activity of DC. This enzyme is thought to be required for maturation and development of sperm cells. Presented in part at the 1988 Joint Meeting of the North Dakota and South Dakota Academies of Science, Bismarck, ND, April 30, 1988. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.     

Heat stability of carboxypeptidase R of experimental animals

Carboxypeptidase N (CPN) and carboxypeptidase R (CPR) are present in fresh serum, and cleave C-terminal arginine or lysine residues from bioactive peptides such as anaphylatoxins and kinins resulting in regulation of peptide activity. Although CPN is present in the active form in plasma, CPR is generated from proCPR by trypsin-like enzymes such as thrombin. CPR regulates not only inflammatory peptides but also restricts fibrinolysis. To elucidate the complex role of CPN and CPR in vivo, studies in animal models will be essential. CPR of guinea pig, rat and rabbit decayed at 37 C rapidly as in the case of human CPR. However, at 25 C, CPR of those species decayed to some extent, although human serum CPR did not decay within 60 min. In the presence of thrombin inhibitor, CPR in the sera of animals tested decayed more rapidly than CPR in serum without thrombin inhibitor suggesting that additional generation of CPR may have been prevented during decay evaluation. However, human serum CPR decayed more rapidly in the absence of thrombin inhibitor indicating that thrombin may accelerate the decay in human serum.