共查询到20条相似文献,搜索用时 15 毫秒
1.
Zöllner A Kagawa N Waterman MR Nonaka Y Takio K Shiro Y Hannemann F Bernhardt R 《The FEBS journal》2008,275(4):799-810
The human 11beta-hydroxylase (hCYP11B1) is responsible for the conversion of 11-deoxycortisol into the major mammalian glucocorticoid, cortisol. The reduction equivalents needed for this reaction are provided via a short electron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-containing reductase. On the biochemical and biophysical level, little is known about hCYP11B1 because it is very unstable for analyses performed in vitro. This instability is also the reason why it has not been possible to stably express it so far in Escherichia coli and subsequently purify it. In the present study, we report on the successful and reproducible purification of recombinant hCYP11B1 coexpressed with molecular chaperones GroES/GroEL in E. coli. The protein was highly purified to apparent homogeneity, as observed by SDS/PAGE. Upon mass spectrometry, the mass-to-charge ratio (m/z) of the protein was estimated to be 55 761, which is consistent with the value 55 760.76 calculated for the form lacking the translational initiator Met. The functionality of hCYP11B1 was analyzed using different methods (substrate conversion assays, stopped-flow, Biacore). The results clearly demonstrate that the enzyme is capable of hydroxylating its substrates at position 11-beta. Moreover, the determined NADPH coupling percentage for the hCYP11B1 catalyzed reactions using either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approximately 75% in both cases. Biacore and stopped-flow measurements indicate that hCYP11B1 possesses more than one binding site for its redox partner adrenodoxin, possibly resulting in the formation of more than one productive complexes. In addition, we performed CD measurements to obtain information about the structure of hCYP11B1. 相似文献
2.
Purification and characterization of human H-ras proteins expressed in Escherichia coli. 总被引:28,自引:10,他引:18
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M Gross R W Sweet G Sathe S Yokoyama O Fasano M Goldfarb M Wigler M Rosenberg 《Molecular and cellular biology》1985,5(5):1015-1024
The full-length normal and T24 mutant human H-ras proteins and two truncated derivatives of the T24 mutant were expressed efficiently in Escherichia coli. The proteins accumulated to 1 to 5% of total cellular protein, and each was specifically recognized by anti-ras monoclonal antibodies. The two full-length proteins as well as a carboxyl-terminal truncated derivative (deleted for 23 amino acid residues) were soluble upon cell lysis and were purified to 90% homogeneity without the use of denaturants. In contrast, an amino-terminal truncated ras derivative (deleted for 22 amino acid residues) required treatment with urea for its solubilization. The guanine nucleotide binding activity of these four proteins was assessed by a combination of ligand binding on proteins blots, immunoprecipitation, and standard filter binding procedures. The full-length proteins showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The showed similar binding kinetics and a stoichiometry approaching 1 mol of GTP bound per mol of protein. The carboxyl-terminal truncated protein also bound GTP, but to a reduced extent, whereas the amino-terminal truncated protein did not have binding activity. Apparently, the carboxyl-terminal domain of ras, although important for transforming function, does not play a critical role in GTP binding. 相似文献
3.
Preparation and characterization of human interleukin-5 expressed in recombinant Escherichia coli. 总被引:2,自引:0,他引:2
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A E Proudfoot D Fattah E H Kawashima A Bernard P T Wingfield 《The Biochemical journal》1990,270(2):357-361
The gene coding for human interleukin-5 was synthesized and expressed in Escherichia coli under control of a heat-inducible promoter. High-level expression, 10-15% of total cellular protein, was achieved in E. coli. The protein was produced in an insoluble state. A simple extraction, renaturation and purification scheme is described. The recombinant protein was found to be a homodimer, similar to the natural murine-derived protein. Despite the lack of glycosylation, high specific activities were obtained in three 'in vitro' biological assays. Physical characterization of the protein showed it to be mostly alpha-helical, supporting the hypothesis that a conformational similarity exists among certain cytokines. 相似文献
4.
A plasmid containing the human HSP70 gene was used to transfect and express the protein in Escherichia coli. The bacterial product was a fusion protein containing 640 amino acids of HSP70, plus 33 additional NH2 terminal amino acids; 12 from the bacterial expression vector and 21 from a 5' human sequence that is not normally translated. It was partially purified by ion-exchange and ATP-Sepharose affinity column chromatography. The bacterially produced human HSP70 protein was then compared with HSP70 obtained from cultured 293 cells. Both shared the same staphylococcal V8 protease peptide fragment pattern, ATP binding, and a weak ATPase activity (about 10-15 nmol ATP hydrolyzed per milligram protein per minute at 30 degrees C). The bacterially produced human HSP70 protein differed in its V8 protease pattern with an E. coli ATP-binding protein that corresponded in molecular mass to the E. coli dnaK gene product. Mutants in the human HSP70 gene were constructed which significantly reduced a predicted major alpha-helical domain in the HSP70 molecule that has partial homology to an ATP-binding site of several protein kinases. One HSP70 mutant clone contained a deletion of 20% at the NH2 terminus, and expressed a 57-kDa product, while the other was missing the middle 50% of the gene (40-kDa product). Neither protein fragment bound to an ATP affinity column, suggesting that ATP binding to HSP70 may be conformationally affected by a region about 20% internal to the NH2 terminal end of the molecule. Recently, a similar location of the ATP-binding site has been reported by Milarski and Morimoto (27). 相似文献
5.
The catalytic and hinge domain (Tyr112-Ile318) of the human membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14), containing hexa-histidines at the C-terminus (chMT1-MMP), was overexpressed in Escherichia coli. The expressed polypeptide was almost exclusively found in the inclusion body, and then purified by a single Ni2+-NTA agarose column chromatography after solubilization with 6 M urea. During refolding, the 26.9 kDa chMT1-MMP was processed to a 24.3 kDa intermediate form and then to a 22.2 kDa mature form. By Western blot analysis and mass spectrometry combined with N-terminal sequencing, the intermediate form was identified as a mixture of the Tyr112-Thr299 with a translation-initiating methionine and Ile114-Thr299, and that the mature form corresponds to Ile114-Pro290. These results demonstrate that the mature form was generated by successive autoproteolysis of the N- and C-terminal sites between Thr299-Thr300, Ala113-Ile114, and Pro290-Thr291 during refolding. Catalytic activity of the mature chMT1-MMP was demonstrated by a peptide cleavage assay. In addition, it has gelatinolytic activity and is able to activate proMMP-2 to the mature MMP-2. These results indicate that the refolded chMT1-MMP retains characteristics of MT1-MMP. 相似文献
6.
Human stromelysin is a member of the matrix metalloproteinase family involved in connective tissue degradation. The stromelysin catalytic domain (SCD) lacking both propeptide and C-terminal fragment was expressed in Escherichia coli in soluble and insoluble forms. The insoluble SCD was refolded to the active form in high yield. The protein showed remarkable thermal stability and was able to cleave a thiopeptolide substrate and its natural substrate proteoglycan. The stable and active 20-kDa protein provides an opportunity to elucidate the structure as well as the mechanism of catalysis and inhibition for matrix metalloproteinases. 相似文献
7.
Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli. 总被引:2,自引:0,他引:2
G N Cox D Pratt D Smith M J McDermott R W Vanderslice 《Protein expression and purification》1999,17(1):26-32
CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells. CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells. Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells. A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo. Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success. Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure. Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay. 相似文献
8.
9.
The side-chain asymmetry of physiological porphyrins is produced by the cooperative action of hydroxymethylbilane synthase and uroporphyrinogen (uro'gen) III synthase. Although the role of uro'gen III synthase is essential for the chemistry of porphyrin biosynthesis, many aspects, structural as well as mechanical, of uro'gen III synthase have yet to be studied. We report here an expression system in Escherichia coli and a purification procedure for human uro'gen III synthase. The enzyme in the lysate was unstable, but we found that glycerol prevents the activity loss in the lysate. The purified enzyme showed remarkable thermostability, particularly when kept in phosphate buffer containing DTT or EDTA, indicating that the enzyme activity may depend on its oxidation state. Examination of the relationship between the number of Cys residues that are accessible to 5,5'-dithiobis(2-nitrobenzoic acid) and the remaining activity during heat inactivation showed that a particular Cys residue is involved in activity loss. From the crystal structure of human uro'gen III synthase [Mathews et al. (2001) EMBO J. 20, 5832-5839], this Cys residue was considered to be Cys73, which is buried deep inside the enzyme, suggesting that Cys73 of human uro'gen III synthase plays an important role in enzyme activity. 相似文献
10.
Purification and characterization of recombinant human interleukin-29 expressed in Escherichia coli 总被引:3,自引:0,他引:3
Human interleukin (IL)-29 is the latest member of the class II cytokine family. However, as a result of lacking efficient method to generate relatively large quantity of IL-29, little is known of its functions in man. In the present study, an Escherichia coli expression system for the rapid expression of the human IL-29 gene was developed. It involved of cloning IL-29 gene into the pET-44 Ek/LIC vector, which allowed expression of IL-29 with a fusion tag consisting of the NusA protein, polyhistidine and S peptide (Nus-His-S-tag), and introducing a thrombin recognition site between the fusion tag and IL-29. The expressed fusion protein was purified by S-protein agarose affinity chromatography, and the fusion tag was removed from recombinant IL-29 by cleavage with thrombin. The purified IL-29 appeared a single band on SDS-PAGE, and the yield of IL-29 was 60 mg from 1 l of bacterial culture. N-terminal sequencing confirmed the identity of the purified protein. The recombinant IL-29 showed specific antiviral activity that was comparable to the commercially available IFN alfa-2b preparation. 相似文献
11.
Lectins are a class of proteins with specific carbohydrate-binding properties found in a wide variety of plants and animals. Gramineae lectins are presumably defense-related proteins in plants that exert their effect by binding to N-acetylglucosamine. Barley lectin is a vacuolar protein synthesized with an amino-terminal signal sequence for entering the secretory pathway and a carboxyl-terminal propeptide necessary for proper targeting to the vacuole. To analyze the three-dimensional structure of barley lectin with the carboxyl-terminal extension and to investigate whether the conversion of the prolectin into the mature molecule leads to a conformational change, the precursor and the mature forms of barley lectin were expressed in Escherichia coli. Both proteins accumulated in denatured form in inclusion bodies were solubilized in 8 M urea and renatured in a redox buffer system. Active pro- and mature barley lectins were purified to homogeneity by affinity chromatography. 相似文献
12.
L. Perez J. Vega C. Chuay A. Menendez R. Ubieta M. Montero G. Padron A. Silva C. Santizo V. Besada L. Herrera 《Applied microbiology and biotechnology》1990,33(4):429-434
Summary The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.
Offprint requests to: L. Perez 相似文献
13.
Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli 总被引:14,自引:0,他引:14
P Wingfield M Payton J Tavernier M Barnes A Shaw K Rose M G Simona S Demczuk K Williamson J M Dayer 《European journal of biochemistry》1986,160(3):491-497
The high-level expression of human interleukin-1 beta in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1/LAF) assay. The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively. 相似文献
14.
G K Reddy S Gunwar C B Green D T Fei A B Chen S C Kwok 《Archives of biochemistry and biophysics》1992,294(2):579-585
In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin. 相似文献
15.
Refolding and characterization of recombinant human GST-PD-1 fusion protein expressed in Escherichia coli 总被引:3,自引:0,他引:3
Li DW Yu JF Chen YJ Ma HB Wang ZF Zhu YB Zhang XG 《Acta biochimica et biophysica Sinica》2004,36(2):141-146
Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed onactivated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibitsproliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involvedin antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain wasamplified and cloned into expression plasmid pGEX-Sx-3. The fusion protein GST-PD-1 was effectivelyexpressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-formed to obtain bioactive soluble GST-PD-I. Fusion protein of above 95% purity was acquired by a conve-nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependentin vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-Ll and PD-l and increase the production of IL-2 and IFN-γ of phytohemagglutinin-activated T cells. 相似文献
16.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1996,1292(1):168-176
Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively. 相似文献
17.
Mapping of antigenic domains of Sendai virus nucleocapsid protein expressed in Escherichia coli. 总被引:4,自引:3,他引:1
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Several nonoverlapping epitopes were mapped on the primary sequence of the Sendai virus NP protein. After a complete cDNA clone of the Sendai virus NP gene was expressed in Escherichia coli, deletion constructs were used to generate a series of overlapping NP fragments deleted at their C termini. Immunoblot analyses with 11 monoclonal antibodies identified four antigenic sites. All of these sites resided in the C-terminal half of NP and were also the only sites detected with a polyclonal serum. These findings confirm and extend the evidence that the C terminus of the NP protein represents the domain exposed on the surface of the nucleocapsid. One of the monoclonal antibodies reacted with a site, comprising only 6 amino acids, lying with a hinge between an alpha-helix and a beta-strand in the predicted secondary structure of NP. Since this antibody is a potent inhibitor of in vitro viral RNA synthesis (K. L. Deshpande and A. Portner, Virology 139:32-42, 1984), the epitope may be critical to the flexibility of the NP molecule that makes the RNA template accessible during RNA synthesis. 相似文献
18.
As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) has drawn considerable attention. This report presented the purification and characterization ofsoluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized andrefolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purifiedto electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purifiedsTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with EDs0 about 1.5mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had astructure similar to that of native protein with 13-sheet secondary structure. This efficient procedure ofsTRAIL renaturation may be useful for the mass production of this therapeutically important protein. 相似文献
19.
Purification and characterization of a Bacillus polymyxa beta-glucosidase expressed in Escherichia coli.
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The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter. Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression. The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose. Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein. This band was shown by a zymogram to correspond to beta-glucosidase activity. The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene. Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined. It was shown that the enzyme is competitively inhibited by glucose. The effects of different metallic ions and other agents were studied. Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect. Ethanol did not show the stimulating effect observed with other beta-glucosidases. The mechanism of enzyme action was investigated. High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound. Results presented here indicate that this compound is cellotriose formed by transglycosylation. 相似文献
20.
Transferrin (Tf) crystals diffract to only medium resolution. The mediocre quality of the crystals may be due to two factors: (1) the genetic variations naturally present in the primary sequence of Tf, and (2) the glycosylation of the protein. To control genetic variations and glycosylation of samples of Tf, it would be desirable to express the Tf gene from a recombinant clone. Additionally, expression of Tf from a clone would allow for manipulation of the structure of Tf. The cDNA encoding Tf has been cloned into the pL-based expression vector, pRE1, and the T7-based expression vectors, pRSETA and pET11A. The Tf expression plasmids, pTF-SSn and pTF-ESn, based on the T7 expression vectors, efficiently produce a 76-kDa protein that is approximately the same size as deglycosylated Tf, cross reacts with anti-Tf antibodies, and matches the deduced N-terminal amino acid sequence. Expression of Tf in Escherichia coli will allow the production of genetically pure, unglycosylated protein. 相似文献