蛋白质二硫键异构酶——一种可能参与蛋白质生物合成的酶

蛋白质二硫键异构酶分布很广,种属间比较保守,定位于内质网膜上,组织分布、活力水平与含二硫键的蛋白质的合成平行,而且底物专一性很差,催化巯基二硫键交换,提示它可能参与蛋白质的生物合成。     

包涵体蛋白的层析复性技术研究进展

包涵体蛋白的复性是生物工程下游技术中的一个重要难题。层析法用于蛋白质复性是一种较新的、适用于大多数蛋白的方法。其原理是将层析技术应用于蛋白质复性和纯化,使变性蛋白质在层析柱上重折叠为正确的空间构象,在洗脱的同时实现部分纯化。本文详细介绍了蛋白质在5种层析柱上的复性方法、原理、应用及研究的新进展,为层析法对蛋白质复性的进一步应用提供依据。     

蛋白质复性研究进展

许多蛋白在大肠杆菌中高效表达时,其产物常以无活性的包含体形式存在,包含体蛋白的复性往往是制备这些蛋白的关键步骤之一,蛋白复性包括肽链折叠和分子内二硫键的氧化这两个互相影响的过程,本文综述了蛋白折叠过程的研究进展,及促进蛋白折叠和二硫键氧化的方法。     

蛋白质复性技术

重组蛋白质的过表达常导致其在胞内发生错误折叠和聚集,形成被称为包含体的聚集体。因此,蛋白质复性是许多基因重组蛋白质药物生产过程的重要步骤。本文简要介绍包含体提取、纯化和溶解工艺,重点阐述蛋白质复性技术,包括稀释复性、稀释添加剂、人工分子伴侣、柱色谱复性和反胶团溶解复性等。最后展望蛋白质复性技术的发展和应用,特别是荷电介质对同电荷蛋白质复性的促进作用。     

蛋白质二硫键异构酶家族的结构与功能

蛋白质二硫键异构酶（protein disulfide isomerase,PDI）家族是一类在内质网中起作用的巯基-二硫键氧化还原酶.它们通常含有CXXC（Cys-Xaa-Xaa-Cys,CXXC）活性位点,活性位点的两个半胱氨酸残基可催化底物二硫键的形成、异构及还原.所有PDI家族成员包含至少一个约100个氨基酸残基的硫氧还蛋白同源结构域.PDI家族的主要职能是催化内质网中新生肽链的氧化折叠,另外在内质网相关的蛋白质降解途径（ERAD）、蛋白质转运、钙稳态、抗原提呈及病毒入侵等方面也起重要作用.     

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．     

蛋白质二硫键异构酶—一种可能参与蛋白质生物合成的酶

蛋白质二硫键异构酶分布很广,种属间比较保守,定位于内质网膜上,组织分布、活力水平与含二硫键的蛋白质的合成平行,而且底物专一性很差,催化巯基二硫键交换,提示它可能参与蛋白质的生物合成。     

二硫键与蛋白质的结构

二硫键是肽链上2个半胱氨酸残基的巯基基团发生氧化反应形成的共价键．具有链内二硫键和链间二硫键2种形式。与氨基酸的氨基氮原子之间形成的稳定共价键不同．二硫键容易被还原而断裂,断裂后可再次氧化重新形成二硫键,因而是可以动态变化的化学键。二硫键是参与一级结构也是形成高级结构的重要化学键,对蛋白质折叠和高级结构的形成与维持十分重要。讨论了二硫键的形成和特征及其与蛋白质结构和功能之间的关系,并讨论了生物学教学中关于二硫键的一些疑问．     

蛋白质二硫键异构酶的纯化和活力测定

 从500g新鲜牛肝制得蛋白质二硫键异构酶(PDI,EC 5.3.4.1)98mg。该酶制剂在SDS-聚丙烯酰胺凝胶电泳中表现为亚基分子量62,000的均一条带。在260nm追踪,因二硫键错接而失活的牛胰核糖核酸酶A,经PDI作用使其二硫键重排恢复活力,从而催化酵母RNA的水解来测定PDI活力。这种单波长法比文献中介绍的追踪A_(260)—A_(280)的双波长法更为灵敏方便。酶的克分子消光系数ε_M=1.03×10~5(pH7.5),其比活性为1400单位/克蛋白质。     

蛋白质复性技术研究进展

评述了蛋白质复性研究的科学背景及蛋白质折叠机制的研究现状 ,详细介绍近年来蛋白质复性技术的研究进展 ,包括稀释添加技术和各种辅助因子的作用、固定化辅助因子应用、尺寸排阻色谱和固定化辅助因子色谱等。     

Solid-phase refolding of poly-lysine tagged fusion protein of hEGF and angiogenin

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinantE. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solidphase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below 30%, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to theN-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5–3 times to about 88%. Besides the intrinsic affinity of angiogenin to heparin the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, fromc.a 4.8% in the conventional refolding of the untagged fusion protein to 63.6%. The process was highly reproducible. The refolded protein in the column eluate retained R Nase bioactivity, of angiogenin.     

Enhancing multiple disulfide bonded protein folding in a cell-free system

A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction.     

肽合成中多对二硫键的形成策略及分析方法

二硫键的正确配对是富含二硫键多肽合成的关键。本文综述了含两对二硫键以上的多肽二硫键的形成策略,优化方法、以及二硫键配对方式的测定方法。     

Disulfide bonds in a recombinant protein modeled after a core repeat in an aquatic insect's silk protein.

We constructed a gene encoding rCAS, recombinant constant and subrepeat protein, modeled after tandem repeats found in the major silk proteins synthesized by aquatic larvae of the midge, Chironomus tentans. Bacterially synthesized rCAS was purified to near homogeneity and characterized by several biochemical and biophysical methods including amino-terminal sequencing, amino acid compositional analysis, sedimentation equilibrium ultracentrifugation, and mass spectrometry. Complementing these techniques with quantitative sulfhydryl assays, we discovered that the four cysteines present in rCAS form two intramolecular disulfide bonds. Mapping studies revealed that the disulfide bonds are heterogeneous. When reduced and denatured rCAS was allowed to refold and its disulfide bonding state monitored, it again adopted a conformation with two intramolecular disulfide bonds. The inherent ability of rCAS to quantitatively form two intramolecular disulfide bonds may reflect a previously unknown feature of the in vivo silk proteins from which it is derived.     

Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.     

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.     

Probing protein folding and stability using disulfide bonds

Disulfide bonds are required to stabilize the folded conformations of many proteins. The rates and equilibria of processes involved in disulfide bond formation and breakage can be manipulated experimentally and can be used to obtain important information about protein folding and stability. A number of experimental procedures for studying these processes, and approaches to interpreting the resulting data, are described here.     

Chaperone-mediated refolding of recombinant prochymosin

It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.     

Cell-free synthesis of proteins that require disulfide bonds using glucose as an energy source

The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50 microM IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines.     

Peptide disulfide RKCGCFF facilitates oxidative protein refolding by mimicking protein disulfide isomerase

This communication reports a new design of peptide disulfide, RKCGCFF, for facilitating oxidative protein refolding. The new design mimics the properties of protein disulfide isomerase (PDI) by introducing hydrophobic and positively charged patches into the two terminals of disulfide CGC. RKCGCFF was found more effective than the traditional oxidant oxidized glutathione (GSSG) as well as its counterpart, RKCGC, in facilitating the oxidative refolding of lysozyme. More importantly, RKCGCFF could improve lysozyme refolding yield at a high concentration (0.7 mg/mL). The research proved that incorporation of hydrophobic and charged patches into the CGC disulfide made the oxidant more similar to PDI in structure and properties.