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1.
Determination of the crystal structure of human MetAP1 makes it possible, for the first time, to compare the structures of a Type I and a Type II methionine aminopeptidase (MetAP) from the same organism. Comparison of the Type I enzyme with the previously reported complex of ovalicin with Type II MetAP shows that the active site of the former is reduced in size and would incur steric clashes with the bound inhibitor. This explains why ovalicin and related anti-angiogenesis inhibitors target Type II human MetAP but not Type I. The differences in both size and shape of the active sites between MetAP1 and MetAP2 also help to explain their different substrate specificity. In the presence of excess Co(2+), a third cobalt ion binds in the active site region, explaining why metal ions in excess can be inhibitory. Also, the N-terminal region of the protein contains three distinct Pro-x-x-Pro motifs, supporting the prior suggestion that this region of the protein may participate in binding to the ribosome. 相似文献
2.
Two genes in the Escherichia coli genome, ypdE and ypdF, have been cloned and expressed, and their products have been purified. YpdF is shown to be a metalloenzyme with Xaa-Pro aminopeptidase activity and limited methionine aminopeptidase activity. Genes homologous to ypdF are widely distributed in bacterial species. The unique feature in the sequences of the products of these genes is a conserved C-terminal domain and a variable N-terminal domain. Full or partial deletion of the N terminus in YpdF leads to the loss of enzymatic activity. The conserved C-terminal domain is homologous to that of the methionyl aminopeptidase (encoded by map) in E. coli. However, YpdF and Map differ in their preference for the amino acid next to the initial methionine in the peptide substrates. The implication of this difference is discussed. ypdE is the immediate downstream gene of ypdF, and its start codon overlaps with the stop codon of ypdF by 1 base. YpdE is shown to be a metalloaminopeptidase and has a broad exoaminopeptidase activity. 相似文献
3.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1997,1344(2):139-152
Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)6-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/l of culture medium. We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques. The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine. His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles. Lipid-bound native apoA-I and His-apoA-I showed very similar α-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I). The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes. In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values. Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I. The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture. In conclusion, we show that His-apoA-I expressed in E. coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux. 相似文献
4.
Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli 总被引:12,自引:0,他引:12
The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases. 相似文献
5.
Yang G Kirkpatrick RB Ho T Zhang GF Liang PH Johanson KO Casper DJ Doyle ML Marino JP Thompson SK Chen W Tew DG Meek TD 《Biochemistry》2001,40(35):10645-10654
The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested. 相似文献
6.
Characterization of the human calmodulin-like protein expressed in Escherichia coli. 总被引:3,自引:0,他引:3
The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16,800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca(2+)-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same Vmax as normal CaM, albeit with a 7-fold higher Kact. In contrast, the erythrocyte plasma membrane Ca(2+)-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca(2+)-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca(2+)-binding protein showing characteristics of a CaM isoform. 相似文献
7.
Réhault SM Zechmeister-Machhart M Fortenberry YM Malleier J Binz NM Cooper ST Geiger M Church FC 《Biochimica et biophysica acta》2005,1748(1):57-65
The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni(2+)-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3 x 10(4) M(-1) min(-1), respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 mug/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies. 相似文献
8.
Renaturation, purification, and characterization of human truncated macrophage colony-stimulating factor expressed in Escherichia coli 总被引:1,自引:0,他引:1
K Yamanishi M Takahashi T Nishida Y Ohmoto M Takano S Nakai Y Hirai 《Journal of biochemistry》1991,109(3):404-409
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. 相似文献
9.
Structure-activity studies of human IL-1 beta with mature and truncated proteins expressed in Escherichia coli 总被引:2,自引:0,他引:2
J S Lillquist P L Simon M Summers Z Jonak P R Young 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(6):1975-1981
IL-1 beta is synthesized as an inactive 31-kDa intracellular protein, which is then processed upon secretion to an active 17-kDa carboxyl-terminal fragment. To identify the minimal portion of IL-1 beta required for activity, we constructed several deletion mutants of mature IL-1 beta. These included three amino-terminal deletions of 10, 16, and 81 amino acids, two carboxyl-terminal deletions of 17 and 72 amino acids, and one internal fragment between amino acids 17 and 81. Expression of the mutants was monitored by Western blots and immunoprecipitation. With one exception, all of these mutants and the full length 17-kDa IL-1 beta were expressed as soluble protein in Escherichia coli and could be assayed for activity and receptor binding in lysates without further purification. Whereas the intact 17-kDa IL-1 beta retained full biologic activity (greater than 10(7) U/ml of lysate) and competed for binding with 125I-labeled IL-1 beta, none of the lysates containing IL-1 beta deletion mutant proteins had activity or competed for binding to receptor at significantly higher concentrations. The loss of function in the smallest C-terminal deletion mutant does not appear to be due to the direct involvement of these C-terminal residues in receptor binding because both monoclonal and polyclonal antisera directed to this region bind to IL-1 beta but do not neutralize its activity. Therefore, this region is probably indirectly involved in sustaining the structure of the receptor-binding site. 相似文献
10.
A truncated v-abl-derived tyrosine-specific tyrosine kinase expressed in Escherichia coli.
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M L Pritchard D Rieman J Feild C Kruse M Rosenberg G Poste R G Greig B Q Ferguson 《The Biochemical journal》1989,257(2):321-329
Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity. Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M). 相似文献
11.
Propionyl-CoA carboxylase (PCC) is a biotin-dependent mitochondrial enzyme that catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA. PCC consists of two heterologous subunits, alpha PCC and beta PCC, which are encoded by the nuclear PCCA and PCCB genes, respectively. Deficiency of PCC results in a metabolic disorder, propionic acidemia, which is sufficiently severe to cause neonatal death. We have purified three PCCs containing pathogenic mutations in the beta subunit (R165W, E168K, and R410W) and one PCCB polymorphism (A497V) to homogeneity to elucidate the potential structural and functional effects of these substitutions. We observed no significant difference in Km values for propionyl-CoA between wild-type and the variant enzymes, which indicated that these substitutions had no effect on the affinity of the enzyme for this substrate. Furthermore, the kinetic studies indicated that mutation R410W was not involved in propionyl-CoA binding in contrast to a previous report. The three mutant PCCs had half the catalytic efficiency of wild-type PCC as judged by the kcat/Km ratios. No significant differences have been observed in molecular mass or secondary structure among these enzymes. However, the variant PCCs were less thermostable than the wild-type. Following incubation at 47 degrees C, blue native-PAGE revealed a lower oligomeric form (alpha2beta2) in the three mutants not detectable in wild-type and the polymorphism. Interestingly, the lower oligomeric form was also observed in the corresponding crude Escherichia coli extracts. Our biochemical data and the structural analysis using a beta PCC homology model indicate that the pathogenic nature of these mutations is more likely to be due to a lack of assembly rather than disruption of catalysis. The strong favorable effect of the co-expressed chaperone proteins on PCC folding, assembly, and activity suggest that propionic acidemia may be amenable to chaperone therapy. 相似文献
12.
Nemoto TK Ohara-Nemoto Y Ono T Kobayakawa T Shimoyama Y Kimura S Takagi T 《The FEBS journal》2008,275(3):573-587
V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His(6)-tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I. 相似文献
13.
Recombinant human midkine (rh-midkine) was expressed under the control of the AOX1 gene promoter in Pichiapastoris. Approximately 640 mg of rh-midkine was secreted into one liter of medium of the high cell-density fermentation. The protein processing of the rh-midkine was done efficiently and correctly in P. pastoris, and O-mannosylation was not detected in the purified rh-midkine. However, only about 30% of the purified rh-midkine was intact. The other ones lost 5-12 amino acid residues from the amino-termini, provably by proteolysis. Even the mixture of these truncated midkines could promote CHO cell proliferation as well as the authentic rh-midkine. 相似文献
14.
Hardern IM Knauper V Ernill RJ Taylor IW Cooper KL Abbott WM 《Protein expression and purification》2000,19(2):246-252
We describe here the expression of a C-terminally truncated form of human procollagenase-3 in Escherichia coli. The protein was found almost exclusively in inclusion bodies that were solubilized and refolded by two separate methods and then purified on Ni-NTA agarose. The purified proenzyme could be activated with either trypsin or APMA and active enzyme could be purified on a peptidic hydroxamate affinity column. Competitive elution from the affinity matrix yielded a highly purified preparation. 相似文献
15.
Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations. 总被引:43,自引:30,他引:13
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Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell. The integration reaction is mediated by integrase, a viral enzyme. Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity. Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates. Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration). Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities. Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region. All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein. However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA. This hypothesis is supported by the results of disintegration assays performed with altered substrates. The results support a model involving separate viral and target DNA-binding sites on integrase. 相似文献
16.
The precursor of bovine adrenodoxin (pAd), a mitochondrial protein, was expressed in Escherichia coli. The cloned cDNA of pAd was ligated to an expression vector pET-3d, and silent mutations were introduced into the N-terminal portion of the cDNA in order to increase the expression. The precursor was highly expressed (approximately 20% of the total cell protein) as the inclusion body, and contained an iron-sulfur center as judged from its optical absorption spectra. The inclusion body was solubilized with 7 M urea and pAd was purified in the presence of urea. The purified pAd was efficiently imported into isolated bovine adrenal cortex mitochondria and processed to the mature form. The import reaction required ATP inside the mitochondria in addition to the inner membrane potential, and was strongly inhibited by trypsin treatment of the mitochondria, as in the case of the in vitro translated precursor. It was, however, not dependent on the unfolding activity of the cytosolic factor with extramitochondrial ATP. 相似文献
17.
Two genes coding for chloramphenicol acetyltransferase and human interferon gamma, respectively, were overexpressed constitutively in two different strains of Escherichia coli (E. coli LE392 and E. coli XL1). The N-terminal amino acid analysis of the purified proteins showed that: (a) the N-terminal methionine is processed more efficiently in E. coli LE392 rather than in E. coli XL1 cells; (b) the N-terminal methionine is removed better from the heterologous human interferon gamma in comparison with the homologous chloramphenicol acetyltransferase protein: and (c) there is no strong correlation between the efficiency of N-terminal procession and the yield of recombinant protein. 相似文献
18.
Intrinsic fluorescence of a truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli 总被引:2,自引:0,他引:2
A M Gilles H Munier T Rose P Glaser E Krin A Danchin C Pellecuer O Barzu 《Biochemistry》1990,29(35):8126-8130
A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms. 相似文献
19.