蛇毒纤溶酶Fibrolase的凝胶过滤色谱复性研究

目的：研究蛇毒纤溶酶Fibrolase的最佳复性方法。方法：使用透析和凝胶色谱法对Fibrolase复性进行研究,比较2种复性方法的相对复性率及蛋白质回收率。结果：2种复性方法的复性率分别为20％、25％,蛋白质回收率分别为16％、5％。结论：凝胶过滤色谱复性优于透析法复性,凝胶过滤色谱复性使蛋白质的复性与纯化同步进行,简化了操作程序,提高了产品回收率。     

重组蛋白复性技术研究进展

本对近年来重组蛋白复性技术的研究进行了评述。比较分析了液相和固相复性的各种方法,提出了复性优化的方案,介绍了在化学复性基础上发展物理复性如高压复性法的新思路。     

重组羧肽酶原B的复性方法研究*

构建的羧肽酶原B表达质粒在大肠杆菌中获得高表达。但目的蛋白是以包涵体的形式存在。为了获得活性羧肽酶B,必须对其包涵体进行变复性。首先利用稀释复性确定了羧肽酶原B复性的最佳缓冲液;在凝胶过滤复性中,研究了柱长和洗脱流速对羧肽酶原B复性效率的影响;另外对比了稀释复性、透析复性、凝胶层析复性和Ni²⁺亲合层析法等四种方法对羧肽酶原B的复性效果。结果发现,这4种方法的复性效果有以下顺序:凝胶过滤复性>稀释复性>Ni²⁺亲合层析>透析复性。     

重组羧肽酶原B的复性方法研究

构建的羧肽酶原B表达质粒在大肠杆菌中获得高表达。但目的蛋白是以包涵体的形式存在。为了获得活性羧肽酶B,必须对其包涵体进行变复性。首先利用稀释复性确定了羧肽酶原B复性的最佳缓冲液;在凝胶过滤复性中,研究了柱长和洗脱流速对羧肽酶原B复性效率的影响;另外对比了稀释复性、透析复性、凝胶层析复性和Ni2 亲合层析法等四种方法对羧肽酶原B的复性效果。结果发现,这4种方法的复性效果有以下顺序:凝胶过滤复性>稀释复性>Ni2 亲合层析>透析复性。     

蛋白质层析柱复性及工艺评价

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。     

凝胶色谱柱复性在低分子量尿激酶原突变体(DscuPA\|32K)中的应用

将构建的一种具溶栓和抗栓双重功能尿激酶原突变体(DscuPA\|32K)基因,在大肠杆菌中进行表达。由于DscuPA\|32K分子较大并且表达量较高,目的蛋白质基本以包涵体的形式存在。包涵体中的蛋白质是无活性的蛋白质,为了获得有活性的蛋白质,就需要对包涵体进行变性及复性。尝试了一种新的凝胶色谱柱复性方法,并通过柱复性方法与常规的稀释复性方法进行了比较,发现柱复性方法明显优于稀释复性方法,具有成本低,效率高,并对目的蛋白质(DscuPA\|32K)进行了初步纯化等优点,尤其对酶这一类容易失活降解的蛋白质进行复性时,很值得进行推广应用。     

层析辅助体外复性含有多对二硫键的融合蛋白质

为建立一种基于阴离子交换介质辅助的含多对二硫键的抗凝溶栓双功能水蛭素12肽-瑞替普酶融合蛋白质 (HV12p-rPA) 的复性方法,采用Q Sepharose XL作为层析复性介质,通过正交实验考察蛋白质上样量、流速、脲梯度、洗脱液中精氨酸浓度、脲浓度、pH、还原型及氧化型谷胱甘肽等因素对复性过程的影响,探索最佳层析复性条件。结果表明：脲梯度、上样量及精氨酸浓度是影响复性的3个主要因素。脲梯度是复性成功的关键,上样量增大时复性蛋白质比活降低,精氨酸辅助HV12p-rPA复性的最佳浓度为1 mol/L。创建了脲、pH双梯度下的阴离子交换层析辅助HV12p-rPA的复性方法,复性后蛋白质的溶栓比活达到46 520 IU/mg,抗凝比活达到9 980 ATU/mg,与稀释复性方法相比,该方法能使复性蛋白质的溶栓比活提高14~15倍,抗凝比活提高7~8倍。     

人血小板衍生生长因子BB亚型包涵体复性与纯化

目的:优化人血小板衍生生长因子BB亚型(PGDF-BB)包涵体复性方法与纯化条件,获得具有较好生物活性的重组PGDF-BB蛋白。方法:对PGDF-BB包涵体以梯度尿素进行变性,选择最佳包涵体变性浓度;比较不同复性条件下的复性率,稳定PGDF-BB包涵体复性方法;参照该蛋白的理化性质,选择适合PGDF-BB重组蛋白的纯化方法。结果:原核系统内实现了PGDF-BB的高表达;通过优化包涵体复性方法,重组蛋白的包涵体复性率可达40%以上;经过多个纯化方法相结合,PGDF-BB的纯度达到95%。结论:通过实验条件的优化,提高了PGDF-BB包涵体复性率,获得高纯度、高生物活性的重组PGDF-BB蛋白。     

抗胰岛素样生长因子1受体单链抗体复性研究

[目的]建立抗人胰岛素样生长因子1受体单链抗体包涵体复性方法。[方法]首先,在96孔板上进行稀释复性,从72种复性液中筛选最佳条件。每孔复性液2 m L,滴入起始浓度1. 5 mg/m L的包涵体溶解液100μL过夜复性。然后,选择最佳复性液与变性液混合在Superdex 75柱上形成1 cm柱长下降5%的变性液梯度,样品按5%柱体积上样进行柱上复性。[结果]最适稀释复性液为C8(50 mmol/L Tris-Cl,GSH/GSSG=5/0. 5 mmol/L,0. 4 mol/L精氨酸,p H 9.0),对应复性率约为78%;以该复性液为基础通过柱上复性,目标蛋白复性率提高到95%,复性样品抗原结合活性良好。[结论]建立了目标蛋白包涵体柱上复性方法,复性率达到95%,产量达到384 mg/L。     

液胶色谱柱复性在低分子量尿激酶原突变体（DscuPA—32K）中的应用

将构建一种具溶栓和抗栓以重功能尿激酶原突变体（DscuPA-32K)基因,在大肠杆菌中进行表达。由于DscuPA-32K分子较大并且表达量较高,目的的性质基本以包涵体的形式存在。包涵体中的蛋白质是无活性的蛋白质,为了获得有活性的蛋白质,就需要对包涵体进行变性及复性。尝试了一种新的凝胶色谱柱复性方法,并通过柱复性方法与常规的稀释复性方法进行了比较,发现柱复性方法明显优于稀释复性方法,具有成本低,效率高,并对目的的蛋白质（DscuPA-32K)进行了初步纯化等优点,尤其对酶这一类容易失活降解的蛋白质进行复性时,很值得进行推广应用。     

Refolding strategies for ketosteroid isomerase following insoluble expression in Escherichia coli

Dilution and column-based protein refolding techniques are compared for refolding Delta 5-3-ketosteroid isomerase (KSI) with a C-terminus his6-tag. Column refolding was performed by removing the denaturant while the protein was adsorbed in an immobilized metal affinity chromatography column. Both dilution refolding and a single-step column-based refolding strategy were optimized to maximize the recovery of KSI enzyme activity, and achieved refolding yields of 87% and 70% respectively. It was found that the column-based refolding yield was reduced at higher adsorbed protein concentrations. An elution gradient with increasing imidazole concentration was used to selectively elute the biologically active KSI protein following column refolding, with high molecular weight KSI aggregates retained in the column. An iterative column-refolding process was then developed to denature and refold protein retained in the column, which significantly increased the refolding yield at high-adsorbed protein concentrations. Repetition of the column refolding operation increased the refolding yield from 50% to 75% for protein adsorbed at a concentration of 2.9 mg/mL of adsorbent. Although for the KSI protein column-based refolding did not improve the overall refolding yield compared to dilution refolding, it may still be advantageous due to the ease of integration with purification operations, increased control over the refolding conditions, and the ability to segregate refolded protein from inactive aggregates during elution.     

Synergistic coordination of polyethylene glycol with ClpB/DnaKJE bichaperone for refolding of heat‐denatured malate dehydrogenase

The use of polyethylene glycol (PEG) as a refolding additive to a refolding cocktail comprising the molecular bichaperone ClpB and DnaKJE significantly enhances chaperone‐mediated refolding of heat‐denatured malate dehydrogenase (MDH). The critical factor to affect the refolding yield is the time point of introducing PEG to the refolding cocktail. The refolding efficiency reached approximately 90% only when PEG was added at the beginning of refolding reaction. The synergistic coordination of an inexpensive refolding additive PEG with the ClpB/DnaKJE bichaperone system may provide an economical route to further enhance the efficacy of ClpB/DnaKJE refolding cocktail approach, facilitating its implementation in large‐scale refolding processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Influence of solvent conditions on refolding of bovine serum albumin

The effect of solvent conditions on the refolding of bovine serum albumin was studied. The rate and extent of refolding was affected by the type of monovalent salt used in the medium. While NaCl and NaBr promoted refolding, NaClO4 and NaSCN decreased the rate and extent of refolding at 0.2 M concentration. In this respect the relative order in which various anions influenced the refolding process followed the lyotropic series Cl-, Br-, I-, ClO4-, SCN-. Urea exhibited two opposite effects on the refolding of albumin: whereas at low concentrations urea increased the extent of refolding, at concentrations above 2.0 M the rate and extent of refolding were dramatically decreased. Addition of ethanol to the medium greatly decreased the refolding even at concentrations as low as 4% (v/v). The effects of these various additives on the refolding behavior of serum albumin is interpreted in terms of subtle changes in the structure of water. It is also shown that, while such changes in the solvent structure affected the rate and extent of refolding, they did not affect the pathway of refolding.     

大肠杆菌表达的单链抗体柱复性的研究

对包含体表达的抗乙肝病毒表面抗原（HBsAg）的单链抗体（ScFv）纯化复性进行了探索.尝试了利用金属螯合亲和层析和凝胶层析柱进行柱上在位复性的可行性. 对包含体表达的ScFv进行透析复性与柱复性,比较其相对复性率及蛋白质回收率,发现柱上复性效果优于传统的透析复性.抗HBsAg ScFv经凝胶色谱Sephacyl S-200柱复性的相对复性率为98%, 蛋白质回收率为81%.由于将纯化复性同步进行,简化了操作程序,提高了产品的回收率.     

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids

The effects of various refolding additives, including metal cofactors, organic co‐solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well‐known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion‐containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca²⁺ and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3‐fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea‐containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor‐containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.     

Eudragit S-100对人转化生长因子β1（TGF-β1）复性的促进作用及其机制

本研究主要是考察一种对pH较为敏感的多聚化合物聚丙烯酸树脂Eudragit S-100是否对人转化生长因子β1（Transforwing growth factor,TGF-β1）复性具有促进作用. 将以包涵体形式存在TGF-β1进行变性,并将变性蛋白直接加入到含有不同浓度Eudragit的蛋白复性缓冲液中,采用MTT法、荧光分光光度法、圆二色谱以及高效液相色谱法等方法来比较分析不同浓度Eudragit S-100对变性TGF-β1的复性促进作用.实验结果表明,在Eudragit S-100作用下TGF-β1的复性产率比普通稀释复性法显著增高且最高达到53%,研究还表明Eudragit S-100的促进蛋白复性的作用是基于Eudragit S-100与TGF-β1发生了特异性的离子结合反应.通过这一反应,Eudragit S-100遮蔽了蛋白多肽间的疏水基团,有效的抑制了蛋白的聚集进而发挥其促复性功能.     

High productivity refolding of an inclusion body protein using pulsed-fed size exclusion chromatography

Protein refolding using size exclusion chromatography (SEC) is advantageous over conventional refolding methods in terms of ease of automation, simultaneous purification capabilities, and the non-adsorptive protein–matrix interaction which eliminates steric constraints. Despite these advantages, the widespread use of SEC refolding remains restricted by low process productivity and product concentration bottlenecks. This study aims to address those limitations and exploit SEC advantages for large-scale refolding applications. Specifically, this study reports the development of a pulsed-fed size exclusion chromatography (PF-SEC) refolding platform, which successfully refolded E. coli-derived α-fetoprotein (AFP) to achieve 53% refolding yield at 0.9 mg/ml AFP refolding concentration. AFP was introduced into the column by pulsed injection to increase feed load, while suppressing concentration-induced aggregation. Refolding was initiated by a urea gradient in the column, where the gradient length could be readily adjusted to complement pulsed feeding patterns. AFP refolding productivity with PF-SEC improved by 8- and 64-fold compared to ion-exchange chromatography refolding and pulsed dilution refolding, respectively, at a fixed refolding concentration. Through a unique integration of pulsed feeding and urea gradient development, this new PF-SEC refolding methodology overcomes ‘productivity and concentration’-related disadvantages inherent in SEC, and will be scalable for large-scale protein refolding applications.     

An Automatic Refolding Apparatus for Preparative-Scale Protein Production

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.     

Artificial chaperone-assisted refolding in a microchannel

Protein refolding using a simple dilution method in a microchannel often led to the formation of protein aggregates, which bound to the microchannel wall, resulting in low refolding yields. To inhibit aggregation and improve refolding yields, an artificial chaperone-assisted (ACA) refolding, which employed detergents and β-cyclodextrin was used. Model proteins, hen egg white lysozyme and yeast α-glucosidase, were successfully refolded in a microchannel. The microscopic observation showed that the ACA method suppressed protein aggregation and facilitated the refolding of lysozyme, whereas significant aggregation was observed when a simple dilution method was employed. The ACA method increased the lysozyme refolding yield by 40% over the simple dilution approach. Similarly, for α-glucosidase, the refolding yield using the ACA method (ca. 50%) was approximately three times compared with the simple dilution method. The ACA refolding method is a suitable approach to use in the refolding of proteins using a microfluidic system.