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蛋白质复性技术研究进展 总被引:1,自引:0,他引:1
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。 相似文献
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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。 相似文献
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蛋白质复性是一个过程,存在中间阶段,此阶段的各种相互作用力决定了蛋白质能否复性。蛋白质复性要求有一定的条件,如pH、温度、离子强度、蛋白质浓度等。另外多种添加剂能促进蛋白质复性,其中包括表面活性剂、低浓度变性剂、分子伴侣蛋白和各种氧化还原对,但对于不同蛋白质,因其结构及理化特性不同,采取不同复性方法,可以使其达到最佳复性效果。 相似文献
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蛋白质复性工艺的研究一直是重组蛋白药物研发领域的热点。稀释复性法和透析复性法的蛋白损失较大、复性得率不理想,而层析柱可以完成复性同时纯化,并能在高蛋白浓度条件下得到较高的复性率,有利于规模放大,是近年来最受关注的复性工艺。就层析柱复性工艺进行了归纳,包括凝胶过滤层析、离子交换层析、亲和层析、疏水相互作用层析的复性,并对其复性原理及各自的优缺点进行了比较分析,最后从复性工艺的有效性、优越性以及对于规模化生产的适用性三个角度论述复性工艺的评价方法。 相似文献
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重组包涵体蛋白质的折叠复性 总被引:49,自引:1,他引:48
冯小黎 《生物化学与生物物理进展》2001,28(4):482-485
综述了减少包涵体形成、包涵体分离和溶解以及包涵体折叠复性的策略及其最新进展 .详细讨论了包涵体蛋白质折叠复性的基本原则、包涵体折叠复性促进剂和包涵体折叠复性方法 相似文献
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分子伴侣在蛋白质折叠中的作用 总被引:2,自引:0,他引:2
分子伴侣主要由三个高度保守的蛋白质家族组成,这三个家族的成员广泛分布于原核和真核细胞中。TCP1复合物是真核细胞细胞溶质内的伴侣蛋白。分子伴侣在蛋白质折叠过程中防止多肽链形成聚集物或无活性结构,提高正确折叠率。本文重点讨论Stress-70家族蛋白质和伴侣蛋白协助蛋白质折叠过程中的协同性以及伴侣蛋白GroEL和GroES的作用机理。 相似文献
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蛋白质折叠与装配成天然状态的机制,过去根据离体复性实验观察认为是自组装,而近几年来的研究表明体内蛋白质的折叠 与装配并非如此,而是常常依赖于其它辅助因子和ATP水解供能,为辅助性组装。这些辅助因子基本可概括为分子内伴侣、酶类和分子伴侣三大类。 相似文献
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Katsuzumi Okumura Hideto Wakayama Yoshiaki Miyake Keiichi Murayama Toshio Miyake Koji Seto 《Bioscience, biotechnology, and biochemistry》2013,77(11):2969-2972
We have isolated four independent genomic DNA fragments encoding a rice storage protein, glutelin, by using its cDNA as a probe. Restriction mapping and sequencing analyses showed that all four clones encode type II mRNA of glutelin and have very similar nucleotide sequences. However, several differences were found among the four clones with respect to the nucleotide sequences of their coding and 5′-upstream regions, some of which confirmed corresponding changes in the amino acid sequences of the encoded proteins. These results indicate that the genes encoding type II mRNA species of glutelin constitute a multi-gene family of closely related members. 相似文献
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One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding
of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and
functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC).
Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography
to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit
of protein purification. It can be easily automated and formatted for high-throughput process. 相似文献
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大肠杆菌高密度发酵以包涵体形式表达融合蛋白Trx-rPA,表达量22%。包涵体蛋白洗涤后经金属螯合层析纯化,纯度达80%以上。经胱氨酸衍生,以脉冲加样形式复性,复性率可高达30%。经ETI-Sepharose纯化,复性的融合蛋白生物活性可达3.5×105IU/mgPr.。融合蛋白可被rEK酶切释放rPA,酶切效率达85%以上。酶切液经IDA-Sepharose和SP-Sepharose层析纯化,rPA纯度达98%以上,生物活性50万IU/mgPr.。1L发酵液经分离、复性及纯化后,可得高纯度rPA300mg以上。 相似文献
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Yanye Feng Ming Zhang Linlin Zhang Ting Zhang Jianfeng Ding Yingping Zhuang Xiaoning Wang Zhong Yang 《PloS one》2012,7(9)
Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production. 相似文献
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Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington''s, Parkinson''s, and Alzheimer''s diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach. 相似文献
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蛋白质的排阻色谱复性的新进展 总被引:3,自引:0,他引:3
外源蛋白在大肠杆菌中高效表达时 ,常常形成不溶的、无活性的包涵体 ,包涵体蛋白的复性是重组蛋白生产过程中的一个技术难题。排阻色谱 (sizeexclusionchromatography ,SEC)用于蛋白复性是一种较新的、适用于任何一种蛋白的方法 ,与常用的稀释复性法相比 ,它能在高的起始蛋白浓度下对蛋白进行复性 ,活性回收率较高 ,同时又能使目标蛋白得到一定程度的纯化。对使用SEC复性的进展进行了评述 ,其内容包括SEC复性的原理及其复性过程中的影响因素 ,并对其未来发展进行了展望。 相似文献
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Len Ito Masaki Okumura Kohsaku Tao Yusuke Kasai Shunsuke Tomita Akiko Oosuka Hidetoshi Yamada Tomohisa Shibano Kentaro Shiraki Takashi Kumasaka Hiroshi Yamaguchi 《The protein journal》2012,31(6):499-503
Protein refolding constitutes a crucial process for recombinant proteins. We report here on the development of a multifunctional refolding additive, glutathione ethyl ester (GSHEE), prepared from a redox reagent glutathione and an amino acid ethyl ester, an aggregation suppressor. Compared to glutathione, GSHEE showed 3.2-fold higher efficiency for the refolding yield of hen egg lysozyme. More importantly, a low concentration of GSHEE is more effective for refolding than conventional additives, such as amino acid ethyl esters by two orders of magnitude. The high potency of GSHEE makes it a candidate for use as a refolding additive for use in conjunction with reduced and denatured proteins. 相似文献
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《Bioscience, biotechnology, and biochemistry》2013,77(12):2668-2674
We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100°C, cryopreservation at -20°C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12°C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82×10-10, 4.35×10-9, 8.98×10-12, and 3.05×10-11, respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation. 相似文献
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大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEKL,主要以包涵体形式存在。包涵体经4mol/L尿素和0.5%TritonX-100洗涤,以6mol/L盐酸胍、100mmol/LDTT溶解,在胱氨酸存在下,以脉冲加样方式复性。融合蛋白复性在6mmol/L胱氨酸存在下、脉冲加量0.03mg/mL和复性终蛋白浓度0.3mg/mL为最佳复性方案。复性的融合蛋白加2mmol/LCaCL2后快速自切。经IDA-Sepharose及Q-Sepharose纯化,rEKL纯度可达95%以上,可高效酶切重组瑞特普酶融合蛋白Trx-rPA。实现了大规模生产rEKL,每升发酵液经复性及纯化后,可得rEKL60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。 相似文献