双点突变核糖核酸酶抑制因子在毕赤酵母中的表达及对抗氧化能力的影响

人胎盘核糖核酸酶抑制因子(HRI)是一种存在于细胞浆中的50 kDa的酸性蛋白质,富含亮氨酸和半胱氨酸。作为胞浆蛋白可保护细胞不受外来的胰RNase的侵袭。HRI有32个半胱氨酸残基,且多数半胱氨酸残基是成对的并在序列上相连。文章用丙氨酸同时取代cys328/cys329,并将此双突变的HRI的cDNA片段构建于质粒pPIC9K,电击转化入毕赤酵母(Pichia pastoris)GS115中,进行分泌型表达。对表达产物进行亲和层析纯化及抗氧化活性检测。实验结果表明,双点突变后的HRI对RNase A的亲和力几乎没有影响,但其抗氧化能力却增加7～9倍。此种抗氧化能力的提高可能是因为在cys328-cys329之间不能形成二硫键而稳定了HRI的三维结构所致。     

The effect of helix-coil transition on backbone 15N NMR relaxation of isolated transmembrane segment (1–36)-bacteriorhodopsin

Protein disulfide isomerase (PDI) is a multifunctional protein of the endoplasmic reticulum, which catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. It consists of four domains designated a, b, b and a. Both a and a domains contain an active site with the sequence motif -Cys-Gly-His-Cys- involved directly in thiol-disulfide exchange reactions. As expected these domains have structures very similar to the ubiquitous redox protein thioredoxin. A low-resolution NMR structure of the b domain revealed that this domain adopts a fold similar to the PDI a domain and thioredoxin [Kemmink, J., Darby, N.J., Dijkstra, K., Nilges, M. and Creighton, T.E. (1997) Curr. Biol., 7, 239–245]. A refined ensemble of solution structures based on the input of 1865 structural restraints shows that the structure of PDI b is well defined throughout the complete protein except for about 10 residues at the C-terminus of the sequence. 15N relaxation data show that these residues are disordered and not part of this structural domain. Therefore the domain boundaries of PDI can now be fixed with reasonable precision. Structural comparison of the PDI b domain with thioredoxin and PDI a reveals several features important for thiol-disulfide exchange activity.     

Analyses of Native Disulfide Bond Formations in the Early Stage of the Folding Process in Mutant Lysozymes Where the Long-Range Interactions in the Denatured State Were Modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.     

以甘油为碳源发酵Pichia pastoris组成型表达人血管生成抑制素

在Pichia pastoris组成型表达外源蛋白中碳源起着重要的调控作用。分别以葡萄糖、甘油、甲醇和油酸为碳源研究它们对摇瓶发酵GS115(pGAP9K-AS)表达hAS的影响。结果如下:油酸(163mg/L),甘油(83mg/L),葡萄糖(76mg/L),甲醇(57mg/L)。根据以上结果,以甘油为碳源在30L生物反应器中进行GS115(pGAP9K-AS)工程菌的高密度发酵。48h后测得hAS的产量为169mg/L。产物hAS具有免疫活性并能抑制bFGF诱导的CAM血管生成和实验小鼠黑色素瘤的生长。治疗12d后,抑瘤率达到90%,统计学分析结果表明hAS治疗组和PBS治疗组小鼠的肿瘤体积呈现显著性差异(P<0.01)。     

杂合抗菌肽CecA-Mag的人工合成及其在Pichia pastoris中的分泌表达

根据抗菌肽天蚕素A(cecropinA,CA)N端第1~7个氨基酸残基,马盖宁(magainin,M)N端第2~12个氨基酸残基,以毕赤酵母偏爱的密码子设计合成了杂合肽CA(1~7)-M(2~12)基因,同载体pPICZα-A连接后转化Pichia pastoris受体菌SMD1168,在醇氧化酶(AOX)启动子调控下,分子量约1.9kDa的CecA-Mag杂合抗菌肽获得表达,抗菌特性研究表明,该表达产物具有广谱抗菌活性,对多数G-菌及G 菌均有较好的抑菌活性。初步抑菌活性测定,显示该杂合肽对金黄色葡萄球菌、耐氨苄青霉素的大肠杆菌及枯草芽孢杆菌有良好的抑杀活性。酸稳定实验显示pH为3.2时仍具有相当高的活性。热稳定性实验显示该杂合肽100℃加热5min后仍具有抑菌活性。这些特点使得重组抗菌肽CecA-mag在疾病防治和动物饲料添加剂等方面显露出很好的应用前景。     

Effect of the structure of the denatured state of lysozyme on the aggregation reaction at the early stages of folding from the reduced form

We previously demonstrated that the hydrophobic clusters present in hen lysozyme under denaturing conditions were disrupted by the mutation of Trp62 to Gly (W62G). In order to examine the effects of the structure of the denatured state of W62G lysozyme on folding, we analyzed the early events in the folding of reduced W62G lysozyme in detail. From the exchange measurements of disulfide bonds using the variants containing a pair of cysteine residues (1SS), it was found that the formation of disulfide bond in the W62G1SS lysozyme was not accompanied by a prominent interaction between amino acid residues, indicating that the disruption of the hydrophobic core led to the random folding at the early stages in the process of folding of the reduced lysozyme. On the other hand, analyses of the oxidative-renaturation of reduced W62G lysozymes, as well as measurements of the extent of aggregation of the reduced and carboxy amido methylated W62G lysozyme, indicated that the formation of an aggregate is more prominent in the reduced W62G lysozyme than in the reduced wild-type lysozyme. Moreover, a lag phase was detected in the oxidative-renaturation of reduced W62G lysozyme, as based on observations of the recovery of activity. The simulation of the folding process indicated that intermediates were present at the early stages in the folding of the reduced W62G lysozyme. These results suggest that the presence of the intermediates was derived from the random folding at the early stages in the folding process of reduced W62G lysozyme due to the disruption of the structure of the denatured state. Folding thus appears to have been kinetically delayed by these processes, which then led to the significant aggregation of reduced lysozyme. Moreover, from the analysis of amyloid aggregation of the reduced lysozymes, it was suggested that the disruption of the residual structure in denatured state by W62G mutation deterred the formation of the amyloid fibrils of lysozyme.     

前导肽介导的蛋白折叠机制及其对脂肪酶影响的研究进展

大多数蛋白质的形成过程主要由合成前体蛋白和合成功能蛋白两个步骤组成.在这个过程中,前导肽能够辅助蛋白质折叠或抑制它的活性.前导肽作为脂肪酶结构中重要的一段多肽链,通常作为分子内分子伴侣来辅助脂肪酶的折叠,同时该序列上包括糖基化位点在内的一些特殊位点,对酶的活性、极端环境稳定性、甲醇耐受性和底物特异性等性质具有重要影响....     

采用混合策略提高葡萄糖氧化酶在毕赤酵母中的表达水平

为了提高葡萄糖氧化酶 (GOD) 在毕赤酵母中的表达水平,提出了甲醇/山梨醇混合碳源诱导和共表达分子伴侣二硫键异构酶 (PDI) 和透明颤菌血红蛋白 (VHb) 两种策略。利用对照菌株X33/pPIC9k–GOD 在5 L发酵罐放大培养时,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活为456 U/mL,比只采用甲醇作为单一碳源诱导时GOD最终酶活提高了20%。利用整合伴侣蛋白菌株X33/pPIC9k-GOD/pPICZ-PDI-VHb在5 L发酵罐进行高密度发酵,采用甲醇/山梨醇混合碳源诱导,GOD最终酶活达到716 U/mL,蛋白浓度为7.4 g/L。研究结果对提高外源蛋白在毕赤酵母中的表达有重要参考价值。     

The role of Dsb proteins of Gram-negative bacteria in the process of pathogenesis

Tertiary and quaternary structures of extracytoplasmic proteins containing more than one cysteine residue often require introduction of disulfide bonds. This process takes place in an oxidative environment, such as the periplasm of Gram-negative bacteria, and is catalyzed by Dsb (disulfide bond formation) proteins. Mutations in dsb genes influence the conformation and stability of many extracytoplasmic proteins. Thus, many pathogens become partially or fully attenuated due to improper folding of proteins that act as virulence factors. This review summarizes the current knowledge on Dsb proteins and their effect on the pathogenicity of Gram-negative bacteria. The potential application of Dsb proteins in biotechnology is also discussed.     

Comparison of intramolecular and intermolecular reactions in protein folding

The intrinsic component of the standard free energy change for the formation of a disulfide bond in a protein molecule is compared to that for an analogous chemical reaction. The former reaction, which represents theintramolecular formation of a disulfide bond in a protein molecule from a cysteine group containing a mixed disulfide bond with glutathione, and a free cysteine residue, is a unimolecular reaction. In contrast, its chemical analogue is a bimolecular reaction, and corresponds to theintermolecular disulfide interchange between a mixed disulfide-bonded compound between a cysteine residue and glutathione, and a free cysteine molecule. The difference in the intrinsic free energy of the above two reactions is estimated by two different approaches. First, a theoretical estimate of the magnitude of the difference in free energy of the two reactions (for a standard state of 1 M) is obtained using a gas-phase statistical thermodynamic approach, which indicates that the intramolecular reaction is energetically favored over its intermolecular counterpart by as much as 15.6 kcal/mole. For comparison, an experimentally derived value is also obtained, using experimental data from a study by Konishi et al. of the regeneration of the protein ribonuclease A (RNase A) from its reduced form by reduced and oxidized glutathiones. The intrinsic component of the free energy change of the intramolecular reaction, as it occurs in the protein molecule, is obtained from such experimental data by accounting explicitly for the free energy change (assumed to be solely an entropy change) pertaining to the conformational changes (ring closure) that the protein molecule undergoes in the course of the reaction. On the basis of the value derived from such an experimental approach, the intramolecular reaction is also energetically more favorable as compared to its intermolecular analogue, but only by a difference of 2.3 kcal/mole (for a standard state of 1 M). The large apparent discrepancy between the two values estimated from the theoretical and experimental approaches is rationalized by the postulation of several additional factors not inherent in the gas-phase theoretical estimate, such as dehydration and intramolecular hydrogen-bonding effects, which can largely compensate for the otherwise favorable energetics of the intramolecular reaction.     

Structural stability of disulfide mutants of basic pancreatic trypsin inhibitor: A molecular dynamics study

The structure and folding of basic pancreatic trypsin inhibitor (BPTI) has been studied extensively by experimental means. We report a computer simulation study of the structural stability of various disulfide mutants of BPTI, involving eight 250-psec molecular dynamics simulations of the proteins in water, with and without a phosphate counterion. The presence of the latter alters the relative stability of the single disulfide species [5–55] and [30–51]. This conclusion can explain results of mutational studies and the conservation of residues in homologues of BPTI, and suggests a possible role of ions in stabilizing one intermediate over another in unfolding or folding processes. © 1996 Wiley-Liss, Inc.     

共表达分子伴侣PDI和Ero1对灰盖鬼伞过氧化物酶在毕赤酵母中表达的影响

来源于灰盖鬼伞长度为1 092 bp的CiP目的基因与AOX1启动子一起整合进酵母染色体基因组中。重组蛋白CiP在酿酒酵母信号肽的引导下成功分泌到胞外,质谱鉴定为目的蛋白,成功在毕赤酵母中表达灰盖鬼伞过氧化物酶(CiP)。将伴侣蛋白内质网氧化还原酶1(Ero1)、二硫键异构酶(PDI)分别单独及同时转入CiP酵母受体菌中,研究它们对CiP在毕赤酵母中表达的影响。结果表明:在摇瓶中,相对于无分子伴侣的菌株,单独整合PDI及同时整合Ero1、PDI菌株的CiP酶活分别提高了2.43和2.62倍,活力达到316 U/m L和340 U/m L。挑选同时整合Ero1、PDI伴侣蛋白的CiP菌株,5 L发酵罐进行高密度发酵,酶活最高达到3 379 U/m L,比摇瓶提高约10倍。本实验结果较目前已报道的1 200 U/m L已是最高水平。     

重组蛋白包涵体的复性研究

重组蛋白在大肠杆菌中的高表达往往形成不可溶、无生物活性的包涵体,需经过变性溶解后,在适当条件下复性形成天然的构象,才可恢复其生物活性．变复性实验是建立在对蛋白质体外折叠机制的了解的基础上．根据近年来对蛋白质折叠机制的认识和重组蛋白包涵体在复性方面的主要进展,论述以下3个方面的内容：1)蛋白质在细胞内的折叠机制;2)蛋白质体外折叠机制;3)蛋白质复性的策略和方法．