精氨酸对牛胰核糖核酸酶氧化复性的时相性抑制作用

精氨酸常在重组蛋白的体外复性中,作为一种小分子添加剂用于抑制蛋白聚集。精氨酸对蛋白复性折叠过程本身的影响尚不清楚。首次以不易聚集的牛胰核糖核酸酶为研究对象,通过飞行质谱检测氧化复性中间体的变化,通过酶学活性检测蛋白活性的恢复过程,观察了精氨酸对氧化复性的直接影响。发现不同浓度精氨酸对牛胰核糖核酸酶的氧化复性有直接的抑制作用。特别在0.5 mol/L浓度时,精氨酸对牛胰核糖核酸酶氧化复性的抑制作用具有独特的时相依赖性:早期复性可持续至4 h,其活性恢复达30%后,晚期复性基本终止。这种抑制特征与脲的抑制作用有明显的不同,精氨酸没有完全抑制关键结构中间体的形成。结果说明,牛胰核糖核酸酶的氧化复性,除经关键结构中间体的主要通路外,还可能存在一个潜在复性通路,它是其氧化复性后期的关键限速通路。该复性通路可以被0.5 mol/L精氨酸完全阻断。     

一种新型贻贝足丝抗氧化蛋白的原核重组表达及功能

贻贝足丝及其足丝蛋白相关研究对于开发新型水下生物粘附剂具有重要的仿生学意义。足丝蛋白在其粘附过程中需要维持一定的还原态,而目前已报道的足丝抗氧化蛋白仅有MFP-6。此前在厚壳贻贝足丝中鉴定到一种新型的富含半胱氨酸和甘氨酸的足丝蛋白质,该蛋白质被命名为Cys/Gly-Rich-Protein（CGRP）,但是CGRP蛋白在足丝中的作用及机制尚不明确。为此,针对CGRP蛋白,在序列分析基础上,利用原核重组表达手段获得其重组蛋白质,采用2,2-联苯基-1-苦基肼基（2,2-diphenyl-1-picryl hydrazyl radical,DPPH）法检测CGRP重组蛋白经不同条件处理后的抗氧化活性。序列分析结果表明,CGRP蛋白含16.5%的半胱氨酸和10%的甘氨酸,其序列中含有两段半胱氨酸位置保守的重复序列,结构预测表明,其优势构象以无规卷曲为主。同源蛋白质搜索结果表明,CGRP蛋白在数据库中尚无高同源性蛋白质存在。通过密码子优化结合原核重组表达策略成功表达出CGRP重组蛋白,所获得的CGRP重组蛋白具有明显的抗氧化活性,且该活性在其半胱氨酸还原后显著增强(0.91±0.05 vs 0.71±0.11, P<0.01)而在半胱氨酸烷基化之后显著下降(0.08±0.03 vs 0.71±0.11, P<0.01),表明CGRP蛋白的抗氧化活性与其序列中半胱氨酸的自由巯基有关。本研究提示,CGRP蛋白是足丝中一种新的具有抗氧化功能的蛋白质,在足丝粘附过程中推测与MFP-6一起参与了富含多巴的足丝粘附蛋白的还原态维持,对贻贝足丝在固化和粘附过程中防止提前粘附具有重要意义。     

精氨酸脱亚氨酶的表达、纯化和活性研究

目的：建立精氨酸脱亚氨酶的高效表达菌种和纯化工艺路线。方法：人工合成编码支原体精氨酸脱亚氨酶（arginine deiminase, ADI）的基因,构建pBV220-ADI原核表达载体,转染大肠杆菌DH5α中并诱导表达目的蛋白,离子交换层析和分子筛层析法纯化目标蛋白。采用体外精氨酸降解试验测定纯化产物活性。结果：成功构建了原核表达载体pBV220-ADI,基因侧序正确。转化大肠杆菌DH5α后筛选到高水平表达目的蛋白的菌株,目标蛋白以包含体形式存在于胞浆内,表达水平超过全菌体蛋白的35%。采用盐酸胍溶解包含体、低温条件下稀释和透析的方法进行复性。顺次采用阳离子交换和凝胶过滤层析对复性液进行纯化,最终获得纯度达到95%的活性产物。活性测定表明,纯化的ADI比活性为80IU/mg。结论：成功构建了ADI的高效表达菌种,建立了目标物质的分离纯化方法。     

猪毛蛋白水解液中精氨酸含量测定

<正> 由盐酸水解猪毛蛋白制胱氨酸后,其 pH 4.8母液(即水解后第一次中和、存放过滤后的滤液。以下简称母液)尚含有相当量的精氨酸,有继续进行综合利用的价值。我们于1974年上半年正式生产精氨酸盐酸盐。为了进一步掌握生产环节,提高得率,改进工艺,就需要对精氨酸缩合前后的母液进行分析。因此,建立一种简便可行的测定母液中精氨酸含量的方法(?)是于生产有指导意义的。由于母液含有其他种类的氨基酸,采用层析、离子交换树脂等方法进行定量分析存在较大的困难。根据精氨酸的特殊颜色反应——坂口反应进行定量测     

人微小纤溶酶原基因的克隆和表达

用PCR方法扩增人微小纤溶酶原(Microplasmingen,mPlg)基因,再与表达载体重组．构造mPlg原核表达质粒并转化大肠杆菌。阳性克隆pSSE-mPlg经温度诱导表达,SDS-PAGE等方法证明表达产物的分子量约为29kDa。占全菌总蛋白的24％左右,并在菌内形成包涵体。经半胱氨酸再氧化法和空气氧化法复性。表达产物r-mPlg经SK作用后显示纤溶活性。同时对蛋白质浓度、复性时间等因素对复性的影响进行了初步探讨。     

固定化分子伴侣GroE促进变性溶菌酶复性的研究

利用重组大肠杆菌表达制备了分子伴侣GroE(GroEL和GroES),研究了GroE以及GroEL辅助变性溶菌酶复性的作用。结果表明,不仅游离GroEL单独作用可使溶菌酶复性收率达到90%以上,而且固定化GroEL亦可有效地促进蛋白质复性,最佳复性温度为37℃,最佳pH值范围为6～8,复性酶的活性收率在85%以上。另外,固定化GroEL可反复回收利用,表明固定化GroEL有可能在实际生物下游过程中得到应用。     

枸橼酸酐对原核表达重组GM－CSF的修饰作用

根据枸橼酸酐对蛋白质中的游离氨基进行化学修饰后可使蛋白质溶解度提高的原理,以大肠杆菌表达的人重组GM-CSF为模型,研究了枸橼酸酐修饰对含凝血酶识别位点的融合蛋白的作用,发现用微量枸橼酸酐修饰的重组GM-CSF变性、复性更容易,溶解度明显提高,并对凝血酶的消化更为敏感,使凝血酶用量降低100倍．GM-CSF活性测定结果证明枸橼酸酐修饰不影响其生物学活性．这些结果为枸橼酸酐修饰法在大肠杆菌表达重组蛋白纯化中的应用提供了新途径．     

包涵体蛋白体外复性的研究进展

外源基因在大肠杆菌中高水平表达时 ,通常会形成无活性的蛋白聚集体即包涵体。包涵体富含表达的重组蛋白 ,经分离、变性溶解后须再经过一个合适的复性过程实现变性蛋白的重折叠 ,才能够得到生物活性蛋白。近年来 ,发展了许多特异的策略和方法来从包涵体中复性重组蛋白。最近的进展包括固定化复性以及用一些低分子量的添加剂等来减少复性过程中蛋白质的聚集 ,提高活性蛋白的产率。     

9种氨基酸对甲醛捕获能力的研究

本文研究了9种氨基酸对甲醛的捕获效果,筛选出了对甲醛捕获效果较好的4种氨基酸,即精氨酸、赖氨酸、半胱氨酸盐酸盐、组氨酸,并研究了这4种氨基酸在不同温度和时间下对甲醛的捕获规律。结果表明,半胱氨酸盐酸盐的甲醛捕获效果最好,捕获率可达100％,而且受温度影响最小;其次是精氨酸、赖氨酸、组氨酸,捕获效果均在50％左右,但受温度影响较大;精氨酸、赖氨酸在低温下对甲醛生成有促进作用,高温下有捕获作用;组氨酸在高温下对甲醛的捕获率显著提高,可达87．99％。     

包含体的体外复性研究进展

在大肠杆菌中大量表达的重组蛋白质常常形成无活性的、不溶性的包含体。包含体经过液固分离、洗涤、变性溶解后,经过一个合理的复性过程可重新折叠成有活性的蛋白质。本文概述了常用的包含体复性方法,并对近年来出现的色谱复性法和应用一些低分子量添加剂等来提高蛋白质复性的产率做了简述。     

Renaturation of human proinsulin--a study on refolding and conversion to insulin

The production of human proinsulin in Escherichia coli usually leads to the formation of inclusion bodies. As a consequence, the recombinant protein must be isolated, refolded under suitable redox conditions, and enzymatically converted to the biologically active insulin. In this study we describe a detailed in vitro renaturation protocol for human proinsulin that includes native structure formation and the enzymatic conversion to mature insulin. We used a His(8)-Arg-proinsulin that was renatured from the completely reduced and denatured state in the presence of a cysteine/cystine redox couple. The refolding process was completed after 10-30 min and was shown to be strongly dependent on the redox potential and the pH value, but not on the temperature. Refolding yields of 60-70% could be obtained even at high concentrations of denaturant (3M guanidinium-HCl or 4M urea) and protein concentrations of 0.5mg/ml. By stepwise renaturation a concentration of about 6 mg/ml of native proinsulin was achieved. The refolded proinsulin was correctly disulfide-bonded and native and monomeric as shown by RP-HPLC, ELISA, circular dichroism, and analytical gel filtration. Treatment of the renatured proinsulin with trypsin and carboxypeptidase B yielded mature insulin.     

Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration

The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.     

Expression of the activity of cystine/glutamate exchange transporter,system x(c)(-), by xCT and rBAT

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.     

Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli

A cDNA clone encoding the human cysteine protease cathepsin L was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at alkaline pH. 38-kDa procathepsin L was purified by gel filtration at pH 8.0, and a 29-kDa form of the enzyme was purified by gel filtration after autoprocessing of the proenzyme at pH 6.5. The kinetic properties of the 29-kDa species of recombinant cathepsin L were similar to those published for the human liver enzyme (Mason, R. W., Green, G. D. J., and Barrett, A.J. (1985) Biochem. J. 226, 233-241), using benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide as substrate. However, the stability of the recombinant enzyme, and its pH optimum for this substrate was shifted to a higher pH. Structure-function studies of cathepsin L were performed by constructing mutations in either the propeptide portion or the carboxyl-terminal light chain portion of the protein. These constructions were expressed in the E. coli system, and enzymatic activities were assayed following solubilization, renaturation, and gel filtration chromatography of the mutated proteins. Deletions of increasing size in the propeptide resulted in large proportional losses of activity, indicating that the propeptide is essential for proper enzyme folding and/or processing in this renaturation system. Deletion of part of the light chain containing a disulfide-forming cysteine residue or a single amino acid substitution of alanine for this cysteine residue resulted in almost complete loss of activity. These data suggest that the disulfide bond joining the heavy and light chains of cathepsin L is essential for enzymatic activity.     

Functional Importance of Arginine 64 in Chlamydomonas reinhardtii Phosphoribulokinase

Phosphoribulokinase (EC 2.7.1.19) was investigated in wild-type Chlamydomonas reinhardtii and in mutant strains deficient in this enzyme activity. Immunoblot analysis revealed substantial amounts of phosphoribulokinase in mutant 12-2B but none in mutant F-60. The pH optimum of the wild-type enzyme was 8.0 and that of the 12-2B enzyme was 6.5. The mutant kinase possessed a K_m value for ribulose 5-phosphate of about 45 millimolar, nearly three orders of magnitude greater than the wild-type value of 56 micromolar. K_m values for ATP in the range of 36 to 72 micromolar were observed with both wild-type and mutant enzymes. The V_max of the wild-type enzyme was about 450 micromoles per minute per milligram of protein, and values for the mutant enzyme were 140 micromoles per minute per milligram at pH 6.5 and 36 micromoles per minute per milligram at pH 7.8. Thermal stabilities of the wild-type and mutant kinases were similar. Sequence analysis of the 12-2B phosphoribulokinase gene revealed a C to T transition that caused an arginine to cysteine change at position 64 of the enzyme. This arginine residue is conserved in phosphoribulokinases from vascular plants, algae, and photosynthetic bacteria and appears to function in binding ribulose 5-phosphate.     

Isolation and characterization of a basic encephalitogenic protein from the central nervous system of sheep.

A basic protein has been purified from sheep brain. The purified protein sedimented in the analytical centrifuge at 56,000 r.p.m. as an homogenous product. This protein induced an allergic encephalitis when injected into guinea pigs. Some physiochemical properties of the protein were studied: the sedimentation coefficient was 1.52 and the molecular weight was 20,000 +/- 2,000, as estimated by electrophoresis in acrylamide gels containing SDS and urea; the specific extinction coefficient (see article) was 6.01 +/- 0.20. The aminoacid composition of the molecule was determined and its most prominent aspects are a high content of arginine and lysine, the presence of a single tryptophan, the total absence of cysteine and cystine and a blocked N-terminal residue. All these properties are very close to those of human and bovine encephalitogenic proteins.     

Arginine catabolism in Aphanocapsa 6308

The catabolic products of arginine metabolism were observed in Aphanocapsa 6308, a unicellular cyanobacterium, by thin layer chromatography of growth media, by limiting growth conditions, and by enzymatic analysis. Of the organic, nitrogenous compounds examined, only arginine supported growth in CO₂-free media. The excretion of ornithine at a concentration level greater than citrulline suggested the existence in Aphanocapsa 6308 of the arginine dihydrolase pathway which produced ornithine, CO₂, NH₄, + adenosine 5-triphosphate. Its existence was confirmed by enzymatic analysis. Although cells could not grow on urea as a sole carbon source a very active urease and subsequently an arginase were also demonstrated, indicating that Aphanocapsa can metabolize arginine via the arginase pathway. The level of enzymes for both pathways indicates a lack of genetic control. It is suggested that the arginase pathway provides only nitrogen for the cells whereas the arginine dihydrolase pathway provides not only nitrogen, but also CO₂ and adenosine 5-triphosphate.Nonstandard Abbreviations CCCP carbonylcyanide mchlorophenyl hydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - CGP cyanophycin granule protein - PS II photosystem II - PSI photosystem I - TLC thin layer chromatography - TCA trichloroacetic acid - DPM disintegrations per min     

Cathepsin B stability,but not activity,is affected in cysteine:cystine redox buffers

In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine- and/or cystine-containing buffers. Cathepsin B activity in cysteine-containing buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzyme's operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathione-containing buffers. When assessed in cysteine:cystine redox buffers (pH 6.0-7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.     

Carrier-mediated Uptake of Arginine and Urea by Volvox carteri f. nagariensis

Volvox carteri f. nagariensis takes up arginine via a high affinity, highly specific carrier, whereas carriers for neutral and acidic amino acids cannot be detected (even in nitrogen-starved cultures). Exogenous arginine is accumulated against a steep concentration gradient and is incorporated into protein with high efficiency, but it is not catabolized to any significant extent and will not serve as a nitrogen source adequate to support growth. Urea is also taken up by a saturable carrier, but several lines of evidence indicate that the arginine and urea carriers are distinct and different. Preexposure to arginine suppresses arginine uptake while stimulating urea uptake. The K_i values observed for reciprocal, competitive inhibition of uptake by arginine and urea are orders of magnitude different from the respective K_m values for uptake. The two uptake systems show entirely different patterns of sensitivity to inhibition by structural analogs. Finally, the V_max values for arginine and urea uptake fluctuate independently (but in a regular pattern) during the asexual life cycle. The fluctuations of urea uptake activity are of considerable magnitude and appear to be linked to key phases of the developmental program.     

An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.