Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.     

Refolding and purification of yeast carboxypeptidase Y expressed as inclusion bodies in Escherichia coli

The genes encoding carboxypeptidase Y (CPY) and CPY propeptide (CPYPR) from Saccharomyces cerevisiae were cloned and expressed in Escherichia coli. Six consecutive histidine residues were fused to the C-terminus of the CPYPR for facilitated purification. High-level expression of CPY and CPYPR-His(6) was achieved but most of the expressed proteins were present in the form of inclusion bodies in the bacterial cytoplasm. The CPY and CPYPR-His(6) produced as inclusion bodies were separated from the cells and solubilized in 6 and 3 M guanidinium chloride, respectively. The denatured CPYPR-His(6) was refolded by dilution 1:30 into the renaturation buffer (50 mM Tris-HCl containing 0.5 M NaCl and 3 mM EDTA, pH 8.0), and the refolded CPYPR-His(6) was further purified to 90% purity by single-step immobilized metal ion affinity chromatography. The denatured CPY was refolded by dilution 1:60 into the renaturation buffer containing CPYPR-His(6) at various concentrations. Increasing the molar ratio of CPYPR-His(6) to CPY resulted in an increase in the CPY refolding yield, indicating that the CPYPR-His(6) plays a chaperone-like role in in vitro folding of CPY. The refolded CPY was purified to 92% purity by single-step p-aminobenzylsuccinic acid affinity chromatography. When refolding was carried out in the presence of 10 molar eq CPYPR-His(6), the specific activity, N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanine hydrolysis activity per milligram of protein, of purified recombinant CPY was found to be about 63% of that of native S. cerevisiae CPY.     

Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris

The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19kOLase, expressed as inclusion bodies, was solubilized with 6M urea and purified by urea-nickel chelate affinity chromatography. The yield of 19kOLase was 16 mg from 400 ml of cultured cells. 19kOLase in 6M urea could be refolded rapidly by dilution with 50mM Tris-HCl (pH 7.8)-10mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of organic solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1muM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.     

Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants

Normal (Gly12) and activated (Val12) Ha-ras proteins were produced in Escherichia coli, and purified to an apparent homogeneity without using any protein denaturants. The purified proteins contained an equimolar amount of GDP. They were stable in the presence of 5 mM Mg2+ and 25% (v/v) glycerol when incubated at 60 degrees C for 5 min. The binding of GDP to the protein was greatly stabilized by Mg2+. In the presence of 10 mM Mg2+, the bound GDP hardly exchanged with external guanine nucleotides, even at 30 degrees C. The exchange reaction was markedly enhanced in the presence of 10 mM EDTA or 120 mM ammonium sulfate. The rate-limiting step of the exchange reaction was the dissociation of the bound GDP from the ras protein, and this step was facilitated 40- to 100-fold by the addition of EDTA or ammonium sulfate. The dissociation rate of the normal (Gly12) ras protein was 2- to 3-fold faster than that of the activated (Val12) protein. The dissociation constants (Kd) for GDP of the normal and activated ras proteins were 1.2 X 10(-8) and 3.1 X 10(-9) M, respectively. The overall turnover rate of GTPase activity of the normal ras protein (10.8 mmol.mol-1.min-1) was about 10-fold higher than that of the activated protein (1.1 mmol.mol-1.min-1) in the absence of Mg2+ (less than 10(-8) M).     

Efficient solubilization, activation, and purification of recombinant Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli

A cytotoxic protein Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli was solubilized in 10 mM HCl. Protein concentration of saturated solution of the recombinant Cry45Aa in 10 mM HCl was about 25 times higher than that in the buffer of previous method (in 50 mM sodium carbonate buffer, pH 10.5, containing 1 mM EDTA, and 10 mM dithiothreitol). The Cry45Aa solubilized in the acidic solution was activated by pepsin as an alternative to proteinase K in the previous method. Cytotoxic activity against CACO-2 cells of the pepsin-treated Cry45Aa was almost identical to the proteinase K-treated protein. The pepsin-treated Cry45Aa was purified by cation-exchange chromatography. The concentration of the purified protein was 539 microg/ml, which was 27-fold higher than that of the activated Cry45Aa by the previously method. The cytotoxic activity of the purified protein was stable in broad pH region (pH 2.0-11.0) for 3 days, and 97% cytotoxic activity remained after incubation at 30 degrees C for 360 min.     

Purification, refolding, and characterization of recombinant LHRH-T multimer

To make the native LHRH immunogenic, a multimer of LHRH interspersed with T non-B peptides (r-LHRH-d2) was expressed as recombinant protein in Escherichia coli. The expression level of the recombinant protein was around 15% of the total cellular protein and it aggregated as inclusion bodies. Inclusion bodies from the bacterial cells were isolated and purified to homogeneity. Instead of high concentrations of chaotropic agents, r-LHRH- d2 was solubilized in 50 mM citrate buffer at pH 3 containing 2 M urea. The protein was refolded by 5-fold dilution (pulsatile) with cold 10 mM citrate buffer at pH 6 in presence of 0.3 M L-arginine. Purification of r-LHRH-d2 was carried out by successive passages on CM-Sepharose column at pH 6.0 which retained extraneous proteins and pH 4.8 at which r-LHRH-d2 bound to the resin. The elution was carried out by using linear salt gradient (0.1-1 M NaCl). The overall yield of the purified r-LHRH-d2 was 40% of the initial inclusion body proteins. The purity and homogeneity were confirmed by a single homogeneous peak on analytical HPLC eluting out at 29.51 min and by single band on SDS-PAGE reactive with polyvalent anti-LHRH antibodies. Mass spectroscopic analysis indicated the protein to be of 16.6 kDa which equals the theoretically expected mass. The N-terminal amino acid analysis of r-LHRH-d2 showed the sequence which corresponded to the designed protein. The CD spectrum of the refolded r-LHRH-d2 showed that the multimer has considerable beta sheet structure like the monomeric LHRH protein.     

Purification and refolding of a novel cancer/testis antigen BJ-HCC-2 expressed in the inclusion bodies of Escherichia coli

BJ-HCC-2 is one of the cancer/testis antigens that may be the most promising targets for tumor immunotherapy. To investigate the expression of BJ-HCC-2 protein in tumor cells and its capacity to elicit CTL response, the recombinant protein of BJ-HCC-2 was expressed in the inclusion bodies in Escherichia coli. The inclusion bodies were solubilized effectively with 0.3% N-lauroyl sarcosine in alkaline buffer. Under this denatured form, the BJ-HCC-2 protein carrying 6x histidine tag was purified with Ni-NTA affinity chromatography in a single step with a purity of over 97%. The yield of the purified protein was about 78%. The purified recombinant protein was refolded in a simple way. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The recovery rate of refolded protein was 92.1%. The renatured protein displayed its immunoreactivity with the antibodies to BJ-HCC-2 protein by Western blotting. This method of protein purification and refolding is easy to manipulate and may be applicable to the hydrophobic proteins that are unable to be purified by other methods.     

Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152)

A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.     

单纯疱疹病毒1型囊膜糖蛋白gD在大肠杆菌中的表达、纯化及其免疫活性的鉴定

目的:在大肠杆菌中表达1型单纯疱疹病毒(HSV-1)囊膜糖蛋白gD,纯化重组蛋白并对其免疫活性进行鉴定。方法:将HSV-1 gD 基因克隆入原核表达载体pET-28b,利用异丙基-B-D-硫代吡喃半乳糖苷(IPTG)诱导重组质粒转化的大肠杆菌,探讨IPTG浓度、诱导时间、诱导温度对重组蛋白表达的影响;盐酸胍裂解变性包涵体,镍柱亲和层析法纯化gD蛋白,并对纯化后的蛋白进行透析复性;Western blot和ELISA检测gD蛋白的免疫活性。结果:酶切和测序结果表明gD基因克隆入pET-28b载体。该重组质粒转化的大肠杆菌经IPTG诱导后重组蛋白主要以包涵体形式存在,大小约40kDa。gD蛋白诱导表达的最佳条件为0.5mmol/L IPTG于37℃诱导8h。镍柱亲和层析法纯化获得的gD蛋白总量为3.1mg/L,透析复性后获得的gD蛋白总量为1.3mg/L,复性率为41.37%。Western blot及ELISA检测表明表达的gD蛋白具有免疫活性。结论:在大肠杆菌中表达并纯化获得具有免疫活性的HSV-1 gD蛋白,为进一步制备HSV-1诊断试剂和预防疫苗奠定了基础。     

Cloning, purification, and refolding of human paraoxonase-3 expressed in Escherichia coli and its characterization

Human paraoxonase (hPON3) is a high density lipoprotein-related glycoprotein with multi-enzymatic properties and antioxidant activity which is proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. In this study, hPON3 gene was amplified from Human Fetal Liver Marathon-Ready cDNA and expressed in Escherichia coli. A majority of the expressed protein existed as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 and refolded in vitro. The refolded rhPON3 was purified by DEAE-Sepharose Fast Flow and its purity was up to 90%. The Km and Vmax values of refolded rhPON3, in respect to phenylacetate hydrolysis were 7.47 +/- 2.14 mM and 66 +/- 17 U/min/mg (n = 3). The Km and Vmax values of refolded rhPON3, in respect to dihydrocoumarin hydrolysis were 0.83 +/- 0.21 mM and 621 +/- 66 U/min/mg (n = 3). The refolded rhPON3 exhibited similar antioxidant activity to that of rhPON3 purified from the soluble fraction of cell lysate and could effectively protect LDL from Cu2+ induced oxidation.     

Purification of the Caenorhabditis elegans transposase Tc1A refolded during gel filtration chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12-16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Refolding and purification of Apo2L/TRAIL produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli

As a new member of tumor necrosis factor (TNF) superfamily, TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) was produced mainly as inclusion bodies by recombinant Escherichia coli with a temperature-inducible expression system. High concentrations of both biomass (65 g dry cells l(-1)) and inactive TRAIL (4.8 g l(-1)) were obtained by applying a high-cell-density cultivation procedure. After the inclusion bodies were washed and solubilized. TRAIL refolded when at 1 mg ml(-1) by a simple pulse dilution method with a 35% yield. Renatured TRAIL was purified to electrophoretic homogeneity by one-step immobilized metal affinity chromatography. The purified TRAIL showed strong cytotoxicity activity against human pancreatic 1990 tumor cells, with ED50 about 1.6 microg ml(-1).     

还原酶缺陷型大肠杆菌对重组蛋白溶解性的影响

探讨大肠杆菌细胞质氧化还原环境对重组蛋白溶解性的影响。选择含有1对二硫键的牛碱性成纤维细胞生长因子（BbFGF）作为简单蛋白的模式分子,选择含有2对二硫键的人抗HBsAg单链抗体（HBscFv）作为复杂蛋白的模式分子,分别构建表达质粒并转化普通宿主菌和还原酶缺陷型宿主菌E. coli Origami(DE3),比较表达产物的溶解性和纯化产物的活性。结果发现,BbFGF在普通宿主菌中大部分形成包涵体,在Origami(DE3)中为可溶性表达,但表达量降低。两种工程菌的表达产物经离子交换和肝素亲和层析两步纯化后,MTT法测定活性,发现来自还原酶缺陷型宿主菌的BbFGF活性高于普通宿主菌表达产物,二者的ED₅₀分别是1.6 ng/mL和2.2 ng/mL;HBscFv在两种宿主菌中均形成包涵体,包涵体以6 mol/L盐酸胍缓冲液溶解后,镍离子螯合亲和层析纯化并透析复性,间接ELISA测定抗原结合活性,发现二者活性无明显差异,但在Origami(DE3)菌体破碎后的的上清中可检测到HBscFv活性,纯化后产量为1～2 mg/L,而在普通宿主菌破碎后的上清中检测不到HBscFv活性。上述结果说明,改变宿主菌细胞质氧化还原环境对于含有1～2对二硫键的重组蛋白的可溶性表达具有明显促进作用。     

Expression and purification of chicken beta interferon and its antivirus immunological activity

Interferon beta (IFN-β) belongs to a class of natural proteins that can inhibit virus replication. In this paper, nucleic acid sequence encoding chicken interferon beta (chIFN-β) mature protein was cloned and highly expressed in Escherichia coli (E. coli) by 1mM IPTG induction. The expressed chIFN-β was about 20% of total bacterial protein. The recombinant protein in form of inclusion body was then solubilised, refolded and purified to a purity of greater than 95% by immobilized metal ion affinity chromatography (IMAC). This recombinant chIFN-β could inhibit vesicular stomatitis Indiana virus (VSV) and infectious bursal disease virus (IBDV) replication in vitro. Animal experiments showed that the recombinant chIFN-β could decrease pathological lesions caused by the IBDV in bursa of Fabricius. These results will provide useful information for further investigations on veterinary clinical applications and fundamental research.     

Porins of Pseudomonas fluorescens MFO as fibronectin-binding proteins

Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C.     

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.     

On-column refolding purification and characterization of recombinant human interferon-lambda1 produced in Escherichia coli

Interferon-lambda1 (IFN-lambda1) is a member of the recently discovered type III IFNs (IFN-lambda), which possesses antiviral, antitumor, and immunomodulatory activities. In this study, the recombinant human IFN-lambda1 containing a hexahistidine tag was expressed in Escherichia coli. IFN-lambda1 was overexpressed under the control of T7 promoter and most of the protein existed in the form of inclusion bodies. The expressed insoluble protein was solubilized with urea, purified and refolded by one-step immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purified IFN-lambda1 appeared as a single band on SDS-PAGE and the purity was more than 95%. The yield was 86 mg IFN-lambda1 from 1L of bacterial culture. Western blotting and N-terminal sequencing confirmed the identity of the purified protein. The purified IFN-lambda1 exhibited specific antiviral activity as demonstrated by a cytopathic effect reduction assay. Thus, this on-column refolding method provides an efficient way to obtain an active IFN-lambda1 with high yield and high purity.     

重组类胰岛素样生长因子-Ⅰ的纯化与复性

目的 获得高纯度和高活性的胰岛素样生长因子（Insulin-like growth factor, IGF-1）;方法 构建好的BL21大肠杆菌工程菌经IPTG诱导,以融合一段截短型半乳糖苷酶及His-tag形式表达IGF-1融合蛋白(约15,000Da),超声破碎,提取包涵体经镍柱亲和层析后, 用羟氨切割纯化的融合蛋白,纯化后的蛋白质在小分子保护剂及GSH/GSSG的存在下复性。结果 经Ni2＋柱亲和层析, IGF-1纯度达90％以上,复性后得到有较高生物活性的IGF-1。结论 IGF-1发酵及纯化和复性方法的建立为大量生产IGF-1打下了基础。     

Expression, purification, and characterization of recombinant human flotillin-1 in Escherichia coli

Human flotillin-1 (reggie-2), a major hydrophobic protein of biomembrane microdomain lipid rafts, was cloned and expressed in Escherichia coli with four different fusion tags (hexahistidine, glutathione S-transferase, NusA, and thioredoxin) to increase the yield. The best expressed flotillin-1 with thioredoxin tag was solubilized from inclusion bodies, first purified by immobilized metal affinity column under denaturing condition and direct refolded on column by decreasing urea gradient method. The thioredoxin tag was cleaved by thrombin, and the flotillin-1 protein was further purified by anion exchanger and gel filtration column. The purified protein was verified by denaturing gel electrophoresis and Western blot. The typical yield was 3.4 mg with purity above 98% from 1L culture medium. Using pull-down assay, the interaction of both the recombinant flotillin-1 and the native flotillin-1 from human erythrocyte membranes with c-Cbl-associated protein or neuroglobin was confirmed, which demonstrated that the recombinant proteins were functional active. This is the first report describing expression, purification, and characterization of active recombinant raft specific protein in large quantity and highly purity, which would facilitate further research such as X-ray crystallography.     

抑菌蛋白Reg3A的表达纯化与功能

利用原核表达系统表达人源抑菌蛋白Reg3A,经包涵体的复性和纯化获得有体外抑菌功能的活性抑菌蛋白,并对其体外抑菌功能进行初步研究。构建Reg3A原核表达载体PET-32a-Reg3A转化补充稀缺tRNA基因的表达菌株大肠杆菌BL21-Codonplus,阳性重组子采用诱导培养基诱导5h后,采用超声破碎的方法提取包涵体蛋白,经包涵体蛋白的纯化和透析复性后通过Ni-NTA亲和层析交换柱,获得纯度达95%的蛋白质。Western blot鉴定显示在15 kD处有特异性条带。使用纯化后的蛋白进一步进行抑菌圈实验和抑菌活性实验,对获得蛋白的体外抑菌活性进行评估,从而为进一步进行Reg3A蛋白功能的评估及应用奠定基础。