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1.
On the basis of Escherichia coli DNA and vectors pBR322, pUC19, hybrid plasmids restoring Udp+ phenotype in the E. coli deletion (delta udp) mutant have been obtained. The udp gene is carried by a 8 kb PstI fragment (on the pUD2) and by a smaller 2.87 kb PstI-SalGI fragment from the PstI fragment (pUD7). The uridine phosphorylase level was 30 times higher in the cells containing hybrid plasmid as compared to the strain with chromosomal location of the udp gene. On the other hand, the measurements of uridine phosphorylase activity in the cytR- and cya- background indicate that expression of the cloned udp gene escapes partially negative control of the CytR repressor and positive control of cAMP--CRP complex. These data suggest that the 2.87 kb PstI--SalGI-fragment contains the intact udp gene which is transcribed from its own promoter. Increase in the activity of beta-galactosidase encoded by udp-lacZ fusion has been observed in the presence of pUD2 or pUD7, which was suggested to be the consequence of titration of CytR repressor molecules in the operator region of the cloned udp. 相似文献
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Function of serine-52 and serine-80 in the catalytic mechanism of Escherichia coli aspartate transcarbamoylase 总被引:3,自引:0,他引:3
Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme. In particular, oxygens of the phosphate of carbamoyl phosphate interact with Ser-52, Thr-53 (backbone), Arg-54, Thr-55, and Arg-105 from one catalytic chain, as well as Ser-80 and Lys-84 from an adjacent chain in the same catalytic subunit. In order to define the role of Ser-52 and Ser-80 in the catalytic mechanism, two mutant versions of the enzyme were created with Ser-52 or Ser-80 replaced by alanine. The Ser-52----Ala holoenzyme exhibits a 670-fold reduction in maximal observed specific activity, and a loss of both aspartate and carbamoyl phosphate cooperativity. This mutation also causes 23-fold and 5.6-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively. Circular dichroism spectroscopy indicates that saturating carbamoyl phosphate does not induce the same conformational change in the Ser-52----Ala holoenzyme as it does for the wild-type holoenzyme. The kinetic properties of the Ser-52----Ala catalytic subunit are altered to a lesser extent than the mutant holoenzyme. The maximal observed specific activity is reduced by 89-fold, and the carbamoyl phosphate concentration at half the maximal observed velocity increases by 53-fold while the aspartate concentration at half the maximal observed velocity increases 6-fold.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
4.
Smith LE Yang J Goodman L Huang X Huang R Dressman J Morris J Silva RA Davidson WS Cavigiolio G 《Journal of lipid research》2012,53(8):1708-1715
Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies. 相似文献
5.
High expression of a recombinant human calcitonin precursor peptide in Escherichia coli 总被引:4,自引:0,他引:4
Human calcitonin (hCT) is a C-terminus -amidated peptide hormone consisting of 32 amino acids. The amidated structure is essential for its biological activities, and the C-terminal-glycine-extended precursor peptide, hCT[G], is converted to bioactive hCT by a C-terminus--amidating enzyme. An efficient production method is described for the hCT[G] peptide, as a part of the fusion protein consisting of a modified E. coli -galactosidase, linker amino acids and hCT[G]. Stable inclusion bodies of the fusion protein in E. coli were expressed by focusing on the amino acid charge, and the fusion protein was modified by inserting a basic amino acid sequence into its linker region. This modification greatly affected the formation of inclusion bodies. E. coli strain W3110/pG97S4DhCT [G]R4 could produce a large amount of stable inclusion bodies, and the hCT[G] peptide was released quantitatively from the fusion protein by S. aureus V8 protease. This enabled a large-scale production method to be established for the hCT[G] precursor peptide in E. coli to produce mature hCT. 相似文献
6.
Gastrodia anti-fungal protein (GAFP) displays strong inhibitory activity against certain fungal pathogens. Five GAFP analogues with different mutations at mannose-binding sites and the wild-type one were expressed and purified in Escherichia coli. The inhibitory analysis of the purified various GAFPs against the growth of Trichoderma viride indicates that single amino acid mutated-type GAFPs have inhibitory activity, but its activity is much less than the wild-type one. The double and triplicate amino acids mutated GAFPs have very low inhibitory activity. For the first time it was proved that GAFP mannose-binding sites play key role in anti-fungi process. 相似文献
7.
alpha-Synuclein (AS), a major component of Lewy body in Parkinson's disease patients, exists as a natively unfolded protein in physiological buffer. We recently found that the overexpressed AS in Escherichia coli bearing the cloned AS cDNA with no signal sequence was actually located inside the periplasm, but not in the cytoplasm as generally recognized. Therefore, a new protocol for preparing recombinant AS has been developed with only two steps: (1) osmotic shock for release of AS-containing periplasm fraction and (2) ion-exchange chromatography for further purification of AS. By using plasmids and E. coli strains commonly used the new protocol is much more convenient, faster, and cheaper compared to the current methods established since 1994. About 80 mg AS with 95% purity can be regularly prepared from a 1L culture in 3 days. 相似文献
8.
High-level expression and secretion of recombinant mouse endostatin by Escherichia coli 总被引:9,自引:0,他引:9
The expression of murine endostatin was achieved by placing its gene downstream of an alkaline phosphatase gene (phoA) promoter. To ensure proper folding and secretion of the recombinant protein, the mouse endostatin was fused with alkaline phosphatase signal peptide. SDS/polyacrylamide gel electrophoresis analysis of the culture medium of recombinant Escherichia coli cells revealed that endostatin was efficiently secreted. The signal peptide was efficiently cleaved during secretion as demonstrated by N-terminal amino acid sequencing. The maximum yield of secreted endostatin during fermentation was 40 mg/liter. Up to 28 mg of endostatin was purified from 1 liter of cell culture broth. The biological activity of recombinant protein was tested in a bovine aortic endothelial (BAE) cell proliferation assay. The recombinant endostatin inhibited the growth of BAE cells stimulated by basic fibroblast growth factor, and its ED50 was comparable to that from a previous report. Flow cytometric measurements of BAE cells cultivated in medium with endostatin demonstrated a cell cycle arrest mainly in the G0/G1 phase and a decrease in the S phase. 相似文献
9.
Secretion expression of recombinant glucagon in Escherichia coli 总被引:1,自引:0,他引:1
A novel approach for the preparation of recombinant human glucagon was described. An expression vector pAGluT, containing phoA promoter, phoA signal peptide and glucagon gene, was constructed by means of genetic engineering. Escherichia coli strain YK537 was transformed with pAGluT. High-level secretory expression of recombinant human glucagon was achieved. The expression yield of recombinant human glucagon was found to be 80 mg/L, approximately 30% of the total proteins in supernatant. The biological activities and the physicochemical properties of the purified recombinant human glucagon were found to be the same as that of native glucagon. In addition, our results suggested that phoA expression system may be suitable for the expression of other small peptides. 相似文献
10.
Human beta-defensin-2 (hBD2) is a small antimicrobial peptide with potential as a therapeutic agent. The effect of codon usage on the expression of hBD2 in Escherichia coli was studied. Two coding sequences encoding the same hBD2 precursor were both expressed as fusion protein with thioredoxin in E. coli BL21 (DE3). One is the wild-type human cDNA and the other is a gene synthesized by a PCR-based method in which rare codons were altered to those frequently used in E. coli. The expression level of recombinant hBD2 was over 50% of the total cellular protein when the synthetic gene with preferential codons was employed which was a 9-fold enhancement over the wild-type cDNA. The result shows the codon bias of the host was a major barrier in high-level expression of recombinant hBD2 and suggests a similar approach may be used in the expression of other defensins in E. coli. 相似文献
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Increasing the expression yield of active Candida antarctica lipase B (CAL-B) in Escherichia coli was achieved by using a codon-optimized synthetic gene and by mutagenesis to introduce hydrophilic residues on the surface
of CAL-B. Five residues (four leucines and one isoleucine) on the surface of CAL-B were selected and changed with aspartate
after codon optimization. While the codon-optimized synthetic gene of CAL-B did not increase the expression yield, the mutation
increased the activity of the enzyme three-fold (3.3 mg/l of culture) compared to the wild type. The mutant enzyme had similar
hydrolytic activity toward hydrolysis of p-nitrophenyl acetate or p-nitrophenyl butyrate and enantioselectivity toward hydrolysis of (R, S)-1-phenylethyl acetate compared to the wild-type enzyme.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
13.
The effect of temperature on the formation of recombinant protein, apolipoprotein A-IMilano was investigated in the present study. The temperature of the initial growth phase was set at 30°C, while temperature variation in induction phase was arranged in three modes. High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out. Experimental results showed that ApoA-IMilano reached 4.8 g/L with the final cell density of OD600, 150. It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density and the expression of the product. The present study provides a basic procedure for the production of recombinant ApoA-IMilano. 相似文献
14.
Sinsuwan S Yongsawatdigul J Chumseng S Yamabhai M 《Protein expression and purification》2012,83(1):52-58
Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated. 相似文献
15.
R. I. Al-Shekhadat I. V. Dukhovlinov A. I. Kobatov N. A. Klimov A. P. Kozlov 《Applied Biochemistry and Microbiology》2011,47(8):730-736
A simple and efficient method for expression in Escherichia coli cells and purification of a recombinant matrix protein, p17, of human immunodeficiency type I virus has been described. HIV-1
subtype A DNA sequence encoding p17 was obtained by amplification of the viral gag gene segment and cloned into an expression vector under the control of T7Lac promoter. The conditions for cell growth and
induction of p17 synthesis by lactose and its further purification by metal chelate chromatography were optimized. p17 preparations
with 97% purity were obtained; the yield of the protein of 28 mg per 1l of culture was achieved. The obtained protein was
capable of binding antibodies from blood serum of a HIV-infected patient during immunoblotting. 相似文献
16.
Expression of recombinant hemoproteins in Escherichia coli is often limited because a vast majority of the protein produced lacks the heme necessary for function. This is compounded by the fact that standard laboratory strains of E. coli have a limited capacity to withdraw heme from the extracellular environment. We are developing a new tool designed to increase the heme content of our proteins of interest by simply supplementing the expression medium with low concentrations of hemin. This hemoprotein expression (HPEX) system is based on plasmids (pHPEX1-pHPEX3) that encode an outermembrane-bound heme receptor (ChuA) from E. coli O157:H7. This heme receptor, and others like it, confers on the host the ability to more effectively internalize exogenous heme. Transformation of a standard laboratory E. coli protein expression strain (BL-21 [DE3]) with the pHPEX plasmid led to the expression of a new protein with the appropriate molecular weight for ChuA. The receptor was functional as demonstrated by the ability of the transformant to grow on iron-deficient media supplemented with hemin, an ability that the unmodified expression strain lacked. Expression of our proteins of interest, catalase-peroxidases, using this system led to a dramatic and parallel increase in heme content and activity. On a per-heme basis, the spectral and kinetic properties of HPEX-derived catalase-peroxidase were the same as those observed for catalase-peroxidases expressed in standard E. coli-based systems. We suggest that the pHPEX plasmids may be a useful addition to other E. coli expression systems and may help address a broad range of problems in hemoprotein structure and function. 相似文献
17.
High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification. 总被引:4,自引:0,他引:4
Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obese gene coding for leptin was expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140. When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%. 相似文献
18.
Cherentaeva EA Logunov DIu Verkhovskaia LV Mezentseva MV Shmarov MM Ershov FI Naroditskiĭ BS Gintsburg AL 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》2008,(3):37-40
The avian recombinant adenovirus of serotype 1 (CELO) was obtained. The recombinant adenovirus of serotype 1 (CELO) induces expression of human beta-interferon (IB). The expression cassette containing IB gene was placed at the right end of the CELO genome under control of hybrid promoter hEF-1alpha/HTLV. The resulting recombinant adenovirus CELO-IB transduced the avian cell culture LMH. The level of production of the recombinant IB was 0.15 micro/ml. The IB protein yield after affine chromatography purification using Ni-NTA agarose was 50%. The biological activity of the purified IB was high (7.8 x 10(8) MU/microg protein). The purified IB inhibited replication of murine encephalomyocarditis virus (VMEC) in cell culture of human diploid fibroblasts (HDF). Thus, expression system based on avian cell culture is an effective system for producing biologically active protein of human interferon beta. 相似文献
19.
Secretion of recombinant human epidermal growth factor into the periplasm in Escherichia coli cells]
N V Batchikova I B Al'tman S V Lutsenko V A Smirnov I V Nazimov L G Eshkind E A Siniagina A V Azhaev 《Bioorganicheskaia khimiia》1992,18(6):766-776
Secretion vectors were constructed in which a synthetic gene of human epidermal growth factor (hEGF) joined with a gene coding for the leader peptide to one of the E. coli outer membrane major proteins (OmpF) is controlled by tac promoter. The increase of the hEGF yield was achieved by the multiplication of the gene copies. The hEGF in bacterial cells was secreted into periplasm. The recombinant protein was isolated by means of reverse phase chromatography as almost homogenous preparation (greater than 98%), the yield being 7 mg/l bacterial culture. The sequence of twenty-five N-terminal amino acid residues of the isolated hEGF coincided with that of the natural protein. The preparation proved to be biologically active. 相似文献
20.
Convenient modification of the method for oligonucleotide-directed in vitro mutagenesis of cloned DNA 总被引:2,自引:0,他引:2
A new modification of the oligonucleotide-mediated mutagenesis technique has been developed. The proposed methodology has been used to produce specific base changes in the double-stranded plasmid DNA. For this purpose, special cloning vectors have been constructed using the synthetic oligodeoxyribonucleotides. The developed method allows the production of mutant DNA from those of the wild-type with a yield of 10-20%. 相似文献