Analysis of the disulfide bonding pattern between domain sequences of recombinant prochymosin solubilized from inclusion bodies

Prochymosin contains three disulfide bonds linking Cys45 to Cys50, Cys206 to Cys210, and Cys250 to Cys283. To analyze the disulfide bonding pattern between domain sequences in the recombinant prochymosin molecule solubilized from inclusion bodies by 8 M urea (designated as solubilized prochymosin), a simple peptide mapping method was established. This process consists of thiol alkylation, cleavage with cyanogen bromide, diagonal electrophoresis on polyacrylamide gel, and N-terminal sequencing. By using this procedure it was found that Cys45 and Cys50 located in the N-terminal domain are not mispaired with the cysteine residues, located in the C-terminal domain, in the solubilized wild-type prochymosin and its mutants. This result implies that Cys45 and Cys50, the partners of a native disulfide, are restricted in some ordered structures existing in inclusion bodies and remaining after solubilization. These native structural elements act as folding nuclei to initiate and facilitate correct refolding. The strategy of preserving the native-like structures including native disulfide in the solubilized inclusion bodies to enhance renaturation efficiency may be applicable to other recombinant proteins.Both authors contributed equally to this work     

Renaturation and activation of calf prochymosin produced in an insoluble form in Escherichia coli

Calf prochymosin produced in Escherichia coli cells harboring the expression plasmids was insoluble and formed large inclusion bodies, which were solubilized by 8 M urea. The conditions allowing correct refolding of denatured prochymosin were investigated. Dialysis at pH 10 in the presence of 500 mM NaCl was found to give the maximum renaturation, and subsequent acidic treatment for autocatalytic processing of refolded prochymosin allowed almost 100% recovery of chymosin.     

Denaturation studies on natural and recombinant bovine prochymosin (prorennin).

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v.c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.     

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.     

High recovery of prochymosin from inclusion bodies using controlled air oxidation

Refolding of proteins from inclusion bodies is a field of increasing interest for obtaining large amounts of active enzymes. Consequently, the development of inexpensive and scalable processes is required. This is particularly challenging in the case of eukaryotic proteins containing cysteines, which may form disulfide bonds in the native active protein. Previous studies have shown that the formation of disulfide bonds is essential for the refolding of prochymosin. In this work we demonstrate that air oxidation can be efficiently used for the refolding of prochymosin and that 48% of the unfolded protein can be recovered as active enzyme at a final protein concentration of 0.8 mg/ml. Refolding of the protein strictly correlates with the change in pH of the refolding solution. We were able to follow the degree of oxidative renaturation of the prochymosin by simply measuring pH. Thus, the scaling up of the refolding system under controlled conditions was easily achieved. Analyses of different substances as folding aids indicate that the use of L-arginine or neutral surfactants improves the recovery of active protein up to 67% of the initial protein. The overall results indicate that prochymosin can be efficiently and inexpensively refolded with high yields by controlled air oxidation.     

重组人粒细胞集落刺激因子的纯化

重组人粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）在工程菌ｐＣＧ－１／ｒｈＧ－ＣＳＦ／ＤＨ５ａ中以无活性的包涵体形式大量表达。经过菌体破碎分离包涵体、包涵体变性复性后,ｒｈＧ－ＣＳＦ的活性得到恢复。用离子交换和疏水层析纯化了ｒｈＧ－ＣＳＦ,比活性达１．５７×１０８ｕ／ｍｇ,纯度大于９８％。     

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．     

Function of disulflde bond Cys45-Cys50 of prochymosin

The conditions (temperature, time, pH) for solubilizing inclusion bodies of prochymosin mutant, Cys45Asp/Cys50Ser, are identical with those for the wild type. Moreover, they have similar oxidative refolding behavior. Under the same renaturation conditions both of them can undergo correct refolding leading to the formation of activable molecules. This is quite different from the mutant with deletion of Cys250-Cys283, indicating that Cys45-Cys50 contributes less to the correct refolding of prochymosin than Cys250- Cys283. However, deletion of Cys45-Cys50 results in a remarkable decrease of the thermostability of pseudochymosin, suggesting that this disulfide bond plays an important role in stabilizing enzyme conformation. The proteolytic (P) and milk-dotting (C) activities of the mutant of pseudochymosin, Cys45Asp/Cys50Ser, are lower than those of its wild counterpart. The C/P ratio of the former is onefold higher than that of the latter.     

Optimized procedures for purification and solubilization of basic fibroblast growth factor inclusion bodies

The efficiency of purification of basic fibroblast growth factor (bFGF) inclusion bodies using EDTA and nonionic detergents was improved from 25 to 40% by shifting the pH from 8.5 to strong alkaline conditions (pH 9.5 – 10.5). Complete dissolution of bFGF inclusion bodies by guanidinium hydrochloride (> 3 m) was independent of pH and the presence of reducing agents. In contrast, solubilization of bFGF inclusion bodies by urea was pH-dependent and increased in efficiency (e.g. from 0 to 100%) by increasing the pH (from pH 5.0 to 10.5 at 9 m urea). The purification and solubilization procedures are efficient for inclusion body concentrations corresponding to 10 and 100 g per l dry cell weight, respectively.     

Production of Chymosin in Escherichia coli Cells and Its Enzymatic Properties

The production of prochymosin directed by a cloned cDNA under the control of a trp promoter was examined in E. coli C600 and HB101. The latter host exhibited a higher degree of expression as to the production of prochymosin in the form of inclusion bodies, which accounted for more than 15 ~ 20% of the total cellular protein. The conditions for the processing of prochymosin in the inclusion bodies to active chymosin were determined. Several enzymatic properties of the processed bacterial chymosin, such as its specific activities as to milk-clotting and proteolysis, heat stability and Ca^{2 +} dependence of the clotting activity, were almost identical to those of authentic chymosin. However, a slight difference was observed with regard to the immunological reactivity with anti-prochymosin antibody.     

包涵体蛋白体外复性的研究进展

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。     

重组人白细胞介素-6的纯化

重组人白细胞介素－６（ｒｈＩＬ－６）在工程菌ｐＢＶ２２０／ｒｈＩＬ－６／ＤＨ５ａ中以包涵体形式高效表达。ｒｈＩＬ－６经过工程菌体破碎、包涵体分离及抽提、复性、色谱分离后得到高度纯化。纯化产物纯度９５％,具有良好的生物学活性     

Partition features and renaturation enhancement of chymosin in aqueous two-phase systems

Aqueous two-phase systems of polyethylene glycol (molecular mass 1450, 3350 and 6000)-phosphate and polyethylene-polypropylene oxide (molecular mass 8400)-maltodextrin systems were used in order to study the partition features of recombinant chymosin from inclusion bodies. These systems in the presence of 8M urea were used for the solubilization of inclusion bodies containing recombinant chymosin and for the oxidative renaturation of this protein. Recombinant chymosin showed to be partitioned in favour of the top phase in all studied systems with a partition coefficient between 4 and 6. The recovery of the chymosin biological activity was 32% in the polyethylene-polypropylene oxide, while in the polyethylene glycol-phosphate the recovery was 50-59%. The results indicate that the liquid-liquid extraction would be an adequate tool able to isolate and concentrate chymosin from inclusion bodies with a yield of biological activity higher than that obtained from the standard method (43%).     

Improved solubilization of recombinant human growth hormone inclusion body produced in Escherichia coli

Recombinant human growth hormone (r-hGH) overexpressed in Escherichia coli forms inactive and insoluble aggregates as inclusion bodies in the cytoplasm. The efficient solubilization of inclusion bodies is critical for cost-effective production. Contrary to a previous report, in our production system, the solubilization method by alkaline treatment including 2 M urea was ineffective. Hence various buffers containing different concentrations of urea or guanidine hydrochloride (GnHCl) at neutral and alkaline pH were attempted. Efficient solubilization (about 90%) was observed in 100 mM Tris buffer, pH 8.0, with more than 4 M GnHCl, and at pH 12.5 with more than 2 M GnHCl, but not with about 8 M of urea. The r-hGH solubilized at pH 12.5 containing 2 M GnHCl was refolded by simple dilution and purified by DEAE Sepharose anion-exchange chromatography. The biological activity of the resulting r-hGH was comparable with commercially available r-hGH in in vitro cell proliferation assay using the hGH-dependent cell line.     

包涵体复性研究进展(英文)

用基因工程技术在大肠杆菌高水平表达重组蛋白时,通常形成无生物活性的包涵体。包涵体在体外经分离、溶解与重折叠后可实现复性,表现为具有生物活性的蛋白。总结了包涵体的相关复性技术,重点介绍重折叠的最新进展情况 。     

Optimization of the industrial production of the recombinant precursor of human insulin

Conditions were found at the analytical level for the solubilization of a recombinant insulin precursor from inclusion bodies in different buffer systems at a wide pH range in the presence of different reducing (dithiothreitol, dithioerythritol) and chaotropic agents (urea, guanidine hydrochloride) and the subsequent renaturation with the use of redox pairs (cysteine-cystine, oxidized glutathione-reduced glutathione, and others). The scaling of the method for the production of the active substance of genetically engineered human insulin has been performed.     

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.     

Oxidative renaturation of lysozyme at high concentrations

Newly synthesized cloned gene proteins expressed in bacteria frequently accumulate in insoluble aggregates or inclusion bodies. Active protein can be recovered by solubilization of inclusion bodies followed by renaturation of the solubilized (unfolded) protein. The recovery of active protein is highly dependent on the renaturation conditions chosen. The renaturation process is generally conducted at low protein concentrations (0.01-0.2 mg/mL) to avoid aggregation. We have investigated the potential of successfully refolding reduced and denatured hen egg white lysozyme at high concentrations (1 and 5 mg/mL). By varying the composition of the renaturation media, optimum conditions which kinetically favor proper folding over inactivation were found. Solubilizing agents such as guanidinium chloride (GdmCl) and folding aids such as L-arginine present in low concentrations during refolding effectively enhanced renaturation yields by suppressing aggregation resulting in reactivation yields as high as 95%. Quantitatively the kinetic competition between lysozyme folding and aggregation can be described using first-order kinetics for the renaturation reaction and third-order kinetics for the overall aggregation pathway. The rate constants for both reactions have been found to be strongly dependent on denaturant and thiol concentration. This strategy supercedes the necessity to reactivate proteins at low concentrations using large renaturation volumes. The marked increase in volumetric productivity makes this a viable option for recovering biologically active protein efficiently and in high yield in vitro from proteins produced as inclusion bodies within microbial cells. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 221-230, 1997.     

Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosin-containing inclusion bodies.

Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together. We have examined structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin. Prochymosin is a monomeric protein containing three disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analysed by Western blotting of SDS-polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coli.     

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。