Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli

A human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4 x 10(7) units/mg of protein. Peptide mapping of a lysylendopeptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.     

In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein

CapZ is a heterodimeric Ca(2+)-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells. It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length. Here we report the expression of the two muscle-specific isoforms alpha2 and beta1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies. Optimal renaturation conditions were obtained both by simultaneous refolding of urea-solubilized subunits and by rapid dilution into a buffer containing 20% glycerol, 5 mM EGTA, 2 mM DTT, 1 mM PMSF, and 100 mM Tris, pH 7.4. The refolding mixture was incubated for 24 h at 15 degrees C and the protein was concentrated by ultrafiltration. Biochemical characterization of the recombinant heterodimer revealed actin binding activities indistinguishable from those of native CapZ as purified from chicken skeletal muscle. Using the same protocol, we were able to refold the beta1, but not the alpha2 isoform as a single polypeptide, indicating a role for beta1 as a molecular template for the folding of alpha2. The reported recombinant approach leads to high yields of active heterodimer and allows the renaturation and characterization of the beta subunit.     

Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies

We have investigated the refolding and purification of the catalytic domain of human 3',5'-cyclic nucleotide phosphodiesterase 7A1 (PDE7A1) expressed in Escherichia coli. A cDNA encoding an N-terminal-truncated PDE7A1(147-482-His) was amplified by RT-PCR from human peripheral blood cells and inserted into the vector pET21-C for bacterial expression of the enzyme fused to a C-terminal His-tag. The PDE was found to be expressed in the form of inclusion bodies which could be refolded to an active enzyme in buffer containing high concentrations of arginine hydrochloride, ethylene glycol, and magnesium chloride at pH 8.5. The PDE7A1(147-482-His) construct could be purified after dialysis and concentration steps by either Zn2+-IDA-Sepharose chromatography or ResourceQ ion-exchange chromatography to homogeneity. In comparison to the metal-chelate column, the ResourceQ purification resulted in a distinctly better yield and enrichment of the protein. Both the Vmax (0.46 micromol. min(-1). mg(-1) ) and the K(m) (0.1 microM) of the purified enzyme were found to be comparable with published data for native or recombinant catalytically active expressed PDE7A1. Using SDS/PAGE, a molecular mass of 39 kDa was determined (theoretical value 38.783 kDa). As known from several other mammalian PDEs, size-exclusion chromatography using refolded PDE7A1(147-482-His) indicated the formation of dimers. The purified enzyme was soluble at concentrations up to 100 microg/ml. A further increase of protein concentration resulted, however, in precipitation of the enzyme.     

大肠杆菌表达的重组hIL-4的分离纯化

对大肠杆菌表达的重组人白细胞介素 4进行了分离纯化。人 IL- 4基因表达产物在大肠杆菌中以不溶性包含体形式存在 ,经过超声破菌、包含体抽提、洗涤处理 ,可使包含体纯度达 70 %以上 ,用 6mol/L盐酸胍变性溶解包含体沉淀后上清直接稀释复性 ,再经浓缩、透析、离子交换色谱一系列纯化步骤终产物纯度达 95 %以上 ,回收率 1 0 %以上 ,比活性达 2 .5× 1 0 6U/mg     

Hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC) based facile purification of recombinant streptokinase from E. coli inclusion bodies

The downstream processing of recombinant streptokinase (rSK), a protein used for dissolution of blood clots has been investigated employing Escherichia coli inclusion bodies obtained after direct chemical extraction followed by expanded bed adsorption chromatography (EBAC). Streptokinase was over-expressed using high cell density (final OD(600)=40) culture of recombinant E. coli, and an SK protein concentration of 1080 mg l(-1) was achieved. The wet cell pellet after centrifugation was re-suspended in 8M urea containing buffer resulting in direct extraction of almost 97% of cellular proteins into solution. Compared to mechanical disruption using sonication, the direct extraction helped in simultaneous cell lysis and inclusion body (IB) solubilization in a single integrated step. The post-extraction solution containing cell debris and cellular proteins was diluted and directly loaded on to an EBAC column containing Streamline phenyl, without clarification. By passing the solution four times through the column and using 1M NaCl during loading, 82.7% rSK activity could be recovered in the 10mM sodium phosphate buffer used for elution. A 3-fold increase in specific activity of rSK, from 0.18 x 10(5) in cell lysate to 0.53 x 10(5)IU mg(-1) resulted after this step. rSK was further purified to near-homogeneity (specific activity=0.96 x 10(5)IU mg(-1)) by a subsequent ion-exchange step operated in packed bed mode. An overall downstream recovery of 63% rSK was achieved after EBAC and ion exchange chromatography. The paper thus describes the purification of rSK using a three-step regime involving simple, efficient and highly facile steps.     

Renaturation of recombinant proteins produced as inclusion bodies

Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies. Within the last few years specific methods and strategies have been developed to prepare active proteins from these inclusion bodies. These methods include (i) isolation of inclusion bodies after disintegration of cells by mechanical forces and purification by washing with detergent solutions or low concentrations of denaturant, (ii) solubilization of inclusion bodies with high concentrations of urea or guanidine-hydrochloride in combination with reducing reagents, and (iii) renaturation of the proteins including formation of native disulphide bonds. Renatured and native disulphide bond formation are accomplished by (a) either air oxidation, (b) glutathione reoxidation starting from reduced material, or (c) disulphide interchange starting from mixed disulphides containing peptides. The final yield of renatured proteins can be increased by adding low concentrations of denaturant during renaturation.     

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.     

Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.     

Preparation of active recombinant cathepsin K expressed in bacteria as inclusion body

Human cathepsin K (EC 3.4.22.38) is a member of the cysteine protease family with high primary sequence homology to cathepsins S, L, and B. It has been shown that cathepsin K plays a major role in the resorption of the bone matrix by osteoclasts. Cathepsin K has a potential as a drug target for the diseases related to bone matrix metabolism such as osteoporosis. We have expressed recombinant human procathepsin K in Escherichia coli as inclusion bodies. Purified procathepsin K had size of 38kDa which is in agreement with the predicted mass of the construct. Refolding was done by rapid dilution into 50mM Tris-HCl, pH 8.0 buffer containing 5mM EDTA, 10 mM GSH, 1mM GSSG, 0.7 M L-arginine, 0.5 M NaCl, and 1% CHAPS and further dialysis against 25 mM Tris-HCl, pH 8.0 containing 0.5 M NaCl. Mature active cathepsin K was prepared from refolded procathepsin K by incubating at 40 degrees C in pH 4.0 buffers with or without pepsin or cysteine. The presence of pepsin or cysteine in autocatalysis buffer did not have effect on the degree of conversion of nascent to mature cathepsin K, but reduced the autocatalysis time slightly. Proteolytic activity was confirmed using synthetic substrate, and Western blotting identified mature cathepsin K. Active cathepsin K had type I and II collagenolytic activities which could be inhibited by E-64.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.     

Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli

A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR. Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results. The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates.     

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.     

Refolding and purification of non-fusion HPT protein expressed in Escherichia coli as inclusion bodies

The gene encoding hygromycin B phosphotransferase (hpt) is a widely used selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the non-fusion hpt expression plasmid was constructed and introduced into Escherichia coli to produce enough quantity of the HPT protein. High level expressed HPT was achieved but most of the expressed protein aggregated as inclusion bodies. The inclusion bodies were washed, separated from the cells, and solubilized by 0.3% Sarkosyl. The protein was renatured by dilution and dialysis, and then purified by anion-exchange chromatography. The activity is 8 U/mg protein and the purity is about 95%. Further studies showed that the microbially produced HPT protein had comparable molecular weight, immuno-reactivities, N-terminal amino acid sequences, and biological activities with those of the HPT produced by transgenic rice harboring hpt gene. All these results demonstrated the validity of utilizing the microbially produced HPT to assess the safety of the HPT protein produced in genetically engineered rice.     

Antihypertensive activity of recombinant peptide IYPR expressed in Escherichia coli as inclusion bodies

To produce more angiotensin converting enzyme inhibitory peptides (ACEIP), we have established a high-efficiency Escherichia coli expression system. The DNA-coding sequence for the recombinant protein, which was subcloned into the vector pET-30a(+), has been expressed as inclusion bodies in E. coli BL21 (DE3). The influences of induction time and concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) on the expression of recombinant protein were studied. The resulting expression level of the protein accounted for about 31% of cellular protein at a temperature of 37°C, IPTG concentration of 0.6mM and induction time of 7h. The inclusion bodies were washed, separated from the cells, and solubilized with urea. After purification by affinity chromatography, the recombinant protein was recovered with a high purity of about 90%. Molecular weight of the recombinant protein was measured using Tricine-SDS-PAGE. Peptide IYPR was obtained by cleavage of the recombinant protein with trypsin and the IC(50) value was 61 mg/L. The antihypertensive activity in spontaneously hypertensive rats (SHRs) was also investigated. Single oral administration of this peptide in 10-week old SHRs resulted in a significant reduction of systolic blood pressure to 50 mm Hg at 4 h. The data obtained provide a good reference for further development of peptide Ile-Tyr-Pro-Arg into an effective antihypertensive agent for prevention and treatment of hypertension.     

Continuous processing of fusion protein expressed as an Escherichia coli inclusion body

In this study we develop the components of an integrated process for the continuous extraction and purification of a histidine-tagged fusion protein expressed as an inclusion body in Escherichia coli. Lac21 was selected as a model peptide and was expressed as a fusion to ketosteroid isomerase. A purification strategy was developed on a 1-ml batch column before successful scale-up and transfer to a continuous purification system, having a bed volume of 240 ml. Preliminary experiments proved cleavage of the fusion protein. The use of chemical extraction and continuous chromatography gives a flowsheet far superior to the traditional methods for inclusion body processing.     

Expression, purification and characterization of human urodilatin in E. coli

Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.     

豹蛙抗瘤酶的毕赤酵母高效表达、纯化及活性测定

豹蛙抗瘤酶(Onconase,ranpirnase,ONC)对体内外多种肿瘤有很强的杀伤作用,是当前全球重点研究的100种新药之一,为获得高表达与高活性的重组豹蛙抗瘤酶(Recombination onconase,rONC),根据成熟ONC的cDNA序列和毕赤酵母密码子偏好性设计基因并提高其GC含量,分泌信号肽采用酵母α交配因子的pre肽,分别构建表达载体pPIC9/ONC、pPIC9K/ONC和pPICZα-A/ONC,并转染毕赤酵母X-33、GS115和SMD1168,筛选阳性克隆并进行诱导表达。在摇瓶规模筛选最佳载体-宿主组合及优化培养条件之后,进行10 L规模最优培养基的筛选,发酵产物经双水相萃取偶联G50凝胶层析分离纯化。结果表明,pPICZα-A/X-33/ONC组合表达量优于其他组合,且在p H 5.5、23℃条件下诱导7 d,最高表达量达到13 mg/L;在10 L规模条件下,rONC于pH 5.5的低盐基础培养基(Lower basic salt medium,LBSM)、甲醇浓度0.25%条件下诱导7 d,最高表达量为180 mg/L;纯化后的rONC纯度≥95%,收率高于90%;生物活性检测发现,rONC在体外能杀伤多种癌细胞。初步建立了rONC的高效表达与纯化体系,为后续的功能和作用机理研究奠定了一定基础。     

Regulated secretion and purification of recombinant antibodies in E. coli.

A plasmid for optimized protein expression of recombinant Fv antibodies (pOPE) in E. coli was used to express the variable domains of the murine monoclonal antibody HD39 specific for the human B-cell surface antigen CD22. The production of Fv antibodies by pOPE can be regulated over a wide range by varying the IPTG concentration. Antibodies that can discriminate between secreted and nonsecreted Fv antibody fragments were used to show that secretion is the limiting step for the production of functional Fv antibodies. IPTG concentrations above 20 microM increased the total antibody production, but did not yield larger amounts of secreted Fv antibodies. The addition of five histidines to the C terminus facilitates an easy single-step enrichment procedure based on immobilized metal affinity chromatography.