Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

Immobilization of mouse single-chain antibodies for affinity chromatography using the cellulose-binding protein

A laboratory method for obtaining immunoaffinity medium for chromatographic purification of recombinant human interferon alpha2b (IFN-alpha2b) is described. The method is based on oriented and non-covalent immobilization of recombinant antibody fragments on cellulose. The single-chain fragment variable (ScFv) against human IFN-alpha2b was genetically fused to cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome and expressed in Escherichia coli. After the isolation of the target protein in functionally active form from bacteria cells its bioaffinity immobilization on several forms of cellulose powders has been carried out. The crystalline microgranular cellulose with immobilized ScFv-CBD-fusion protein was used as affinity medium to perform the purification of recombinant human IFN-alpha2b directly from clarified extract of E. coli cells.     

Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific beta-glucosidase.

Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)(6)Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)(6)Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after the refolding process was confirmed by K(m) determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.     

特异识别HIV-1序列的锌指蛋白的表达纯化及其在Biacore CM-5芯片上的固定

目的:为了更好地利用Biacore 3000研究锌指与核酸的相互作用,将特异性识别HIV-15′端一段保守序列的三锌指蛋白固定在CM-5芯片上。方法:将特异性识别HIV-15′端一段保守序列5′-CTGTGTTTG-3′的三锌指基因克隆到表达载体pET-22b( )中,转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达重组三锌指蛋白,超声碎菌进行SDS-PAGE分析;包涵体形式的表达产物用盐酸胍溶解后,经一步凝胶柱复性并纯化;随后摸索适宜固定的pH值并通过化学方法进行固定。结果:表达的重组蛋白主要以包涵体形式存在于超声沉淀中,纯化及柱复性后的蛋白纯度为98.8%,并在CM-5芯片上成功固定。结论:本研究为利用Biacore实时定量研究锌指蛋白与其识别DNA的相互作用进行了尝试。     

A Simplified Method for the Efficient Refolding and Purification of Recombinant Human GM-CSF

Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment.     

Penicillin-binding protein 2a of Streptococcus pneumoniae: expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme.

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cell wall. PBPs possess transpeptidase and transglycosylase activities responsible for the final steps of the bacterial cell wall cross-linking and polymerization, respectively. To facilitate our structural studies of PBPs, we constructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-terminal part of the protein including the transmembrane domain) of the pbp2a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli. This GST fusion form of PBP2a, designated GST-PBP2a*, was expressed almost exclusively as inclusion bodies. Using a combination of high- and low-speed centrifugation, large amounts of purified inclusion bodies were obtained. These purified inclusion bodies were refolded into a soluble and enzymatically active enzyme using a single-step refolding method consisting of solubilization of the inclusion bodies with urea and direct dialysis of the solubilized preparations. Using these purification and refolding methods, approximately 37 mg of soluble GST-PBP2a* protein was obtained from 1 liter of culture. The identity of this refolded PBP2a* protein was confirmed by N-terminal sequencing. The refolded PBP2a*, with or without the GST-tag, was found to bind to BOCILLIN FL, a beta-lactam, and to hydrolyze S2d, an analog of the bacterial cell wall stem peptides. The S2d hydrolysis activity of PBP2a* was inhibited by penicillin G. In conclusion, using this expression system, and the purification and refolding methods, large amounts of the soluble GST-PBP2a* protein were obtained and shown to be enzymatically active.     

Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration

The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.     

Disulfide–bridging PEGylation during refolding for the more efficient production of modified proteins

Proteins that are modified by chemical conjugation require at least two separate purification processes. First the bulk protein is purified, and then after chemical conjugation, a second purification process is required to obtain the modified protein. In an effort to develop new enabling technologies to integrate bioprocessing and protein modification, we describe the use of disulfide‐bridging conjugation to conduct PEGylation during protein refolding. Preliminary experiments using a PEG‐mono‐sulfone reagent with partially unfolded leptin and unfolded RNAse T1 indicated that the cysteine thiols underwent disulfide‐bridging conjugation to give the PEGylated proteins. Interferon‐β1b (IFN‐β1b) was then expressed in E.coli as inclusion bodies and found to undergo disulfide bridging‐conjugation during refolding. The PEG‐IFN‐β1b was isolated by ion‐exchange chromatography and displayed in vitro biological activity. In the absence of the PEGylation reagent, IFN‐β1b refolding was less efficient and yielded protein aggregates. No PEGylation was observed if the cysteines on IFN‐β1b were first modified with iodoacetamide prior to refolding. Our results demonstrate that the simultaneous refolding and disulfide bridging PEGylation of proteins could be a useful strategy in the development of affordable modified protein therapeutics.     

Cloning and purification of functionally active Fas ligand interfering protein (FIP) expressed in Escherichia coli

This report presents purification and characterization of the extracellular domain of rat Fas protein, called FIP (FasL interfering protein), expressed as inclusion bodies in Escherichia coli. FIP was extracted from the inclusion bodies, solubilized with 8 M urea, purified by a single-step immobilized metal ion (Ni(2+)) affinity chromatography and refolded. SDS/PAGE and mass spectrometry analysis of the purified protein verified its purity. Fluorescence spectrum analysis showed that the refolding procedure caused structural changes which presumably might have led to oligomerization. The purified FIP has biological activities: it binds specifically soluble Fas ligand and protects human Jurkat lymphocytes against FasL-dependent apoptosis. This efficient procedure of FIP expression in E. coli and renaturation may be useful for production of therapeutically important proteins.     

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.     

One-step Purification and Immobilization in Cellulose of the GroEL Apical Domain Fused to a Carbohydrate-binding Module and its use in Protein Refolding

The apical domain of the chaperonin, GroEL, fused to the carbohydrate binding module type II, CBD_Cex, of Cellulomonas fimi, was expressed in Escherichia coli. The recombinant protein, soluble or from inclusion bodies, was directly purified and immobilized in microcrystalline cellulose particles or cellulose fabric membranes. Assisted refolding of rhodanese by the immobilized mini-chaperone showed a two-fold improvement as compared to a control. Using chromatographic refolding, 35% of rhodanese activity was recovered in only 5 min (mean residence time) as compared to 17% for spontaneous refolding. This mini-chaperone immobilized in cellulose could be a cost-efficient method to refold recombinant proteins expressed as inclusion bodies.     

Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag

A strategy for purification of inclusion body-forming proteins is described, in which the positively charged domain Z(basic) is used as a fusion partner for capture of denatured proteins on a cation exchange column. It is shown that the purification tag is selective under denaturing conditions. Furthermore, the new strategy for purification of proteins from inclusion bodies is compared with the commonly used method for purification of His(6)-tagged inclusion body proteins. Finally, the simple and effective means of target protein capture provided by the Z(basic) tag is further successfully explored for solid-phase refolding. This procedure has the inherited advantage of combining purification and refolding in one step and offers the advantage of eluting the concentrated product in a suitable buffer.     

huGM-CSF(9-127)-IL-6(29-184)融合蛋白的复性及纯化研究

利用Q Sepharose H.P.离子交换柱层析在8mol/L尿素变性条件下对huGM-CSF(9-127)-IL-6(29-184）融合蛋白进行初步纯化,然后再利用Sephacryl S-200分子筛柱层析复性及纯化后获得目的蛋白,其纯度达到95％以上。该纯化方案成功地解决了稀释复性或透析复性产物在进行Q Sepharose H.P.离子交换柱层析时目的蛋白不稳定而沉积于柱上的问题,获得了较好的复性效果,复性率达到80％以上。使用该纯化方案,1天内便可基本完成重组蛋白的复性及纯化过程,而且也便于扩大。     

Direct refolding of inclusion bodies using reversed micelles

The protein refolding of inclusion bodies was investigated using reversed micelles formed by aerosol OT (AOT). Ribonuclease A (RNase A) was overexpressed in Escherichia coli and used as native inclusion bodies. The enzymatic activity of RNase A was completely regained from the inclusion bodies within 14 h by solubilization in reversed micelles. To further enhance the refolding rate, a molecular chaperone, GroEL, was incorporated into the refolding system. The resultant refolding system including GroEL showed better performance under optimized conditions for the refolding of RNase A inclusion bodies. The refolding rate was considerably improved by the addition of the molecular chaperone, and the refolding step was completed in 1 h. The protein refolding in the GroEL-containing refolding system was strongly dependent on the coexistence of ATP and Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active and assisted in the renaturation of the inclusion bodies. The addition of cold acetone to the reversed micellar solution allowed over 90% recovery of the renatured RNase A.     

Production of minichaperone (sht GroEL191-345) and its function in the refolding of recombinant human interferon gamma

The recombinant minichaperone sht GroEL191-345 was cultivated in a 3.7 L stirred bioreactor with the high yield of 216.2 mg/L broth. In the refolding of recombinant human interferon gamma (rhuIFN-gamma) inclusion bodies, more than 2-3 fold enhancement in protein mass recovery and total activity were observed in the presence of free or immobilized minichaperone to the refolding buffer.     

Immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragments

Three foldases--the apical domain of GroEL (mini-chaperone) and two oxidoreductases (DsbA and DsbC) from Escherichia coli--were studied in refolding a protein with immunoglobulin fold (immunoglobulin-folded protein) that had been produced as inclusion bodies in E.coli. The foldases promoted the refolding of single-chain antibody fragments from denaturant-solubilized and reduced inclusion bodies in vitro, and also effectively functioned as alternatives for labilizing agent and oxidizing reagent in the stepwise dialysis system. Immobilization of the oxidoreductases enhanced refolding and recovery of functional single-chain antibody in the dialysis system, suggesting that immobilized oxidoreductases can be used as an effective additive for refolding immunoglobulin-folded proteins in vitro.     

Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli

Overexpression of rhIFN-alpha2b was obtained by synthesizing a codon optimized gene for IFN-alpha2b and expressing it in the form of inclusion bodies (IBs) in Escherichia coli. The recombinant plasmid pRSET-IFNalpha, which had the IFN-alpha2b gene under the T7 promoter, was coexpressed with plasmid pGP1-2, which carried the gene for T7 RNA polymerase under the heat inducible lambdaP(L) promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2g/L at a final OD(600) of 67. At this point, the IBs represented approximately 40% of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of approximately 3g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of approximately 3 x 10(9)IU/mg protein.     

包涵体蛋白的分离和色谱法体外复性纯化研究进展

重组蛋白在大肠杆菌中表达多为无活性的包涵体形式,须经洗涤、溶解、复性后才能得到生物活性蛋白。综述了近年来包涵体蛋白分离纯化和复性技术研究进展,重点讨论了色谱法复性技术的应用,包括尺寸排阻色谱、亲和色谱、离子交换色谱、疏水相互作用色谱、固定化脂质体色谱、扩张床吸附色谱的进展情况。     

重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。