共查询到20条相似文献,搜索用时 46 毫秒
1.
表达的白细胞介素-2-绿脓杆菌外毒素(IL-2-PE)融合蛋白以包含体形式存在于宿主菌中,为分离纯化表达产物提供了方便,但因需进行复性,也增加了后处理的难度.我们采用4mol/L尿素、0.5%TritonX-100的1×PBS洗涤包含体两遍,再经SephacrylS-300分子筛及DEAE-SepharoseFF阴离子交换柱层析后,获得的融合蛋白纯度可达90%~95%。此外,我们从GSSG浓度、L-精氨酸浓度、复性蛋白质的起始浓度、复性液的pH值、复性温度及复性时间等参数入手,系统地研究了融合蛋白的复性条件,探索到了IL-2-ME40和IL-2-PE664Glu融合蛋白复性的最适条件。 相似文献
2.
本文对大肠杆菌表达的重组人白细胞介素-2进行了制备工艺的研究。用4mol/L脲溶解可溶性细菌蛋白后可使rIL—2包涵体纯度达70%;在变性条件下进行凝胶过滤并收集rlL-2主峰纯度可达90%;利用Cu2 氧化复性;最后用反相HPLC纯化,得到高度均一,纯度高达98%,比活达到8.0×106U/mg蛋白,且无热源,无残留DNA的rIL—2;每次反相色谱分离过程可获得400mg左右的rIL-2,得率约25%。 相似文献
3.
4.
以双顺反子表达载体,在大肠杆菌中经IPTG诱导表达了人骨形成蛋白-3羧基端肽段(hBMP-3C),表达量占菌体总蛋白量的18.5%.目的蛋白为25kD、含hBMP-3C端215个氨基酸残基组成的肽段,包括hBMP-3成熟肽和一部分前肽.表达产物以包涵体的形式存在,用含TritonX-100的洗涤液和5mol/L以下脲溶液连续洗涤,可获得较高纯度的重组人骨形成蛋白-3C端肽.经复性处理成可溶性蛋白,植入小鼠肌肉内,第14d组织切片显示有软骨细胞和软骨基质形成,第21d可见成骨细胞和骨基质形成.将rhBMP-3C与脱矿去免疫原性异种骨粒复合后作小鼠肌肉植入试验,21d组织切片上可见硬质骨形成.结果表明:大肠杆菌表达的hBMP-3C经复性后具有诱骨活性,糖基化并非BMP-3活性所必需. 相似文献
5.
外源基因在大肠杆菌中高水平表达时,通常会形成不溶性、无活性的蛋白聚集体即包含体。包含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含体中复性重组蛋白。介绍稀释法、透析及分子排阻、固定化金属离子亲和层析、疏水层析复性等策略和进展;物理化学因素、前导肽协助蛋白折叠;人工分子伴侣辅助蛋白折叠及反胶束、多聚物用于蛋白复性。 相似文献
6.
包涵体蛋白体外复性的研究进展 总被引:38,自引:1,他引:38
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。 相似文献
7.
利用恒溶氧—补料分批技术高密度培养大肠杆菌生产重组人骨形成蛋?… 总被引:10,自引:0,他引:10
对表达人骨形成蛋白-2A的重组大肠杆菌YK537/pDH-B2m在500ml摇瓶中进行了培养条件的摸索实验,继后用5L自控发酵罐进行分批培养和分批补料培养,以获取rhBMP-2A。两种培养方式结果结果比较表明,在培养过程中保持30%-40%左右的溶2解氧和限制性流加葡萄糖可以使BMP-2A的含量达到2.78g/L,最终菌体密度为OD60053,重组蛋白的表达量占菌体总蛋白的25%。 相似文献
8.
利用高效重组表达载体RP66工程菌诱导发酵产生无活性的包体形式重组HIV-1逆转录酶,经过一定条件的摸索,确定了包涵体形RT蛋白洗涤、增溶、复性的条件。复性后的可溶性蛋白,经疏水相互作用和阴离子交换层析进一步分离纯化。 相似文献
9.
高密度培养大肠杆菌YK537/pSBHL—11生产重组人细胞白介素—3 总被引:6,自引:0,他引:6
在B.Brau ES-10型15L和NBS DioFlo3000型5L发酵罐中,利用补料分批培养技术高工表达培养含重组质粒pSBHL-11的大肠杆菌YK537,生产重组人白细胞介素-3(IL-3),发现在发酵过程中,限制性流加甘油,控制溶解氧在30%-40%左右,30℃生长,11h,42℃诱导培养4h,能将发酵液中最终菌体密度从OD16600提高到OD53600,并且保持了白细胞介素-3的表达量。 相似文献
10.
重组人骨形成蛋白—3羧基端肽在大肠杆菌中表达及其诱骨活性 总被引:13,自引:0,他引:13
以双顺反子表达载体,在大肠杆菌中经IPTG诱导表达了人骨形成蛋白-3羧基端肽段(hBMP-3C),表达量占菌体总蛋白量的18.5%。目的蛋白为25kD、含hBMP-3C端215个氨基酸残基组成的肽段,包括hBMP-3成熟肽和一部分前肽。表达产物以包涵体的形式存在,用含TritonX-100的洗涤液和5mol/L以下脲溶液连续涤,可获得较高纯度的重组人骨形成蛋白-3C端肽,经复性处理成可溶性蛋白,植 相似文献
11.
Symmetric primary and tertiary structure mutations within a symmetric superfold: a solution, not a constraint, to achieve a foldable polypeptide 总被引:1,自引:0,他引:1
Brych SR Dubey VK Bienkiewicz E Lee J Logan TM Blaber M 《Journal of molecular biology》2004,344(3):769-780
In previous studies designed to increase the primary structure symmetry within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) a combination of five mutations were accommodated, resulting in structure, stability and folding kinetic properties similar to wild-type (despite the symmetric constraint upon the set of core residues). A sixth mutation in the core, involving a highly conserved Met residue at position 67, appeared intolerant to substitution. Structural analysis suggested that the local packing environment of position 67 involved two regions of apparent insertions that distorted the tertiary structure symmetry inherent in the beta-trefoil architecture. It was postulated that a symmetric constraint upon the primary structure within the core could only be achieved after these insertions had been deleted (concomitantly increasing the tertiary structure symmetry). The deletion of these insertions is now shown to permit mutation of position 67, thereby increasing the primary structure symmetry relationship within the core. Furthermore, despite the imposed symmetric constraint upon both the primary and tertiary structure, the resulting mutant form of FGF-1 is substantially more stable. The apparent inserted regions are shown to be associated with heparin-binding functionality; however, despite a marked reduction in heparin-binding affinity the mutant form of FGF-1 is surprisingly approximately 70 times more potent in 3T3 fibroblast mitogenic assays. The results support the hypothesis that primary structure symmetry within a symmetric protein superfold represents a possible solution, rather than a constraint, to achieving a foldable polypeptide. 相似文献
12.
The biophysical study of protein-protein interactions and docking has important implications in our understanding of most complex cellular signaling processes. Most computational approaches to protein docking involve a tradeoff between the level of detail incorporated into the model and computational power required to properly handle that level of detail. In this work, we seek to optimize that balance by showing that we can reduce the complexity of model representation and thus make the computation tractable with minimal loss of predictive performance. We also introduce a pair-wise statistical potential suitable for docking that builds on previous work and show that this potential can be incorporated into our fast fourier transform-based docking algorithm ZDOCK. We use the Protein Docking Benchmark to illustrate the improved performance of this potential compared with less detailed other scoring functions. Furthermore, we show that the new potential performs well on antibody-antigen complexes, with most predictions clustering around the Complementarity Determining Regions of antibodies without any manual intervention. 相似文献
13.
《Expert review of proteomics》2013,10(1):13-26
Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein–protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein–protein interaction studies. 相似文献
14.
Dimo Kashchiev 《Proteins》2014,82(9):2229-2239
15.
A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone. 相似文献
16.
Paula M. Dalessio Rebecca C. Craven Parvez M. Lokhandwala Ira J. Ropson 《Proteins》2013,81(2):316-325
An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013. © 2012 Wiley Periodicals, Inc. 相似文献
17.
The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25°C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25°C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to ~5%). It is demonstrated that a single universal mathematical function can be used to represent the partial molar heat capacity of the native and unfolded states of proteins in solution. This function can be experimentally written in terms of the molecular weight, the polar and apolar solvent accessible surface areas, and the total area buried from the solvent. This unique function accurately predicts the different magnitude and temperature dependences of the heat capacity of both the native and unfolded states, and therefore of the heat capacity changes associated with folding/unfolding transitions. © 1995 Wiley-Liss, Inc. 相似文献
18.
蛋白质相互作用研究的新技术与新方法 总被引:2,自引:0,他引:2
目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善.在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术.还综述了近两年建立的新方法:与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位-配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法. 相似文献
19.
Protein-fusion constructs have been used with great success for enhancing expression of soluble recombinant protein and as tags for affinity purification. Unfortunately the most popular tags, such as GST and MBP, are large, which hinders direct NMR studies of the fusion proteins. Cleavage of the fusion proteins often re-introduces problems with solubility and stability. Here we describe the use of N-terminally fused protein G (B1 domain) as a non-cleavable solubility-enhancement tag (SET) for structure determination of a dimeric protein complex. The SET enhances the solubility and stability of the fusion product dramatically while not interacting directly with the protein of interest. This approach can be used for structural characterization of poorly behaving protein systems, and would be especially useful for structural genomics studies. 相似文献
20.
Improvement of protein stability in protein microarrays 总被引:1,自引:0,他引:1
Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml–1 to 5 g ml–1 by fluorescence analysis. 相似文献