scholarly journals Overexpression of Soluble Human Thymosin Alpha 1 in Escherichia coli

2005 ◽  
Vol 37 (2) ◽  
pp. 147-151 ◽  
Author(s):  
Pei-Fu Chen ◽  
Hong-Ying Zhang ◽  
Geng-Feng Fu ◽  
Gen-Xing Xu ◽  
Ya-Yi Hou
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soluble Form ◽  
Bacterial Proteins ◽  
Nickel Affinity Chromatography ◽  
Sds Page ◽  
Thymosin Alpha 1

Abstract Synthesized gene of human thymosin alpha 1 (Tα1) was inserted into pET-28a, pET-9c, pThioHis B, pGEX-2T or pBV222 and then inductively expressed in strains of Escherichia coli. Among the five expression systems, the BL21/pET-28a system provides the highest expression level of fusion protein in a soluble form, which is up to 70% of total expressed bacterial proteins as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resulting fusion protein purified through nickel affinity chromatography accounts for 2.53% of the wet bacterial pellet weight and reaches 94.5% purity by SDS-PAGE. These results indicate the potential of this expression system for high-throughput production of recombinant Tα1.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

Isolation and purification of a Campylobacter upsaliensis autolysin

1999 ◽  
Vol 45 (1) ◽  
pp. 23-30
Author(s):  
Somchai Santiwatanakul ◽  
Noel R Krieg
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Micrococcus Luteus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Isolation And Purification ◽  
Whole Cells ◽  
Sds Page ◽  
Autolytic Activity ◽  
Campylobacter Upsaliensis

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS) - PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.Key words: autolysin, Campylobacter upsaliensis, zymogram, murein hydrolase.

Fusion Expression on the Esat-6 and cfp-10 Genes of Mycobacterium bovis in Escherichia coli

2013 ◽  
Vol 421 ◽  
pp. 354-358
Author(s):  
Jia Ming Lin ◽  
Chun Fang Wang ◽  
Jia Ning Guan ◽  
Hong Xia Ma ◽  
Shuang Hou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Bovis ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Single Antigen

Based on the (Gly4Ser)3 linker, theesat-6andcfp-10gene were fused for raising the antigenicity of single antigen. The DNA fragments ofesat-6andcfp-10were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR),and the fusion gene esat-6-cfp-10 were cloned into pMD18-T vector, and then we got the recombinant plasmid pMD-esat-6-cfp-10. pMD-esat-6-cfp-10 and pET28a (+) were digested byBamHI andEcoRI double enzymes. The purified mpb esat-6-cfp-10 fusion gene was subcloned into the expression vector pET28a (+),and the prokaryotic expression vector pET-esat-6-cfp-10 was constructed. Plasmid containing pET-esat-6-cfp-10 was transformed into competenceEscherichia coliBL21(DE3).The bacterium was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE),approximately 25 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western-blotting. The results indicated that the protein was of antigenic activity ofMycobacterium bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of DNA vaccine; living carrier vaccine; subunit vaccine and diagnostic reagents against bovine tuberculosis.

scholarly journals Expression in Escherichia coli of human ARHGAP6 gene and purification of His-tagged recombinant protein.

2003 ◽  
Vol 50 (1) ◽  
pp. 239-247 ◽  
Author(s):  
Anna-Maria Ochocka ◽  
Marzena Czyzewska ◽  
Tadeusz Pawełczyk
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Rho Gtpase ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Step Procedure ◽  
Shock Proteins

In this report we describe cloning and expression of human Rho GTPase activating protein (ARHGAP6) isoform 4 in Escherichia coli cells as a fusion protein with 6xHis. We cloned the ARHGAP6 cDNA into the bacterial expression vector pPROEX-1. Induction of the 6xHis-ARHGAP6 protein in BL21(DE3) and DH5alpha cells caused lysis of the cells irrespective of the kind of culture medium used. Successful expression of the fusion protein was obtained in the MC4100Deltaibp mutant strain lacking the small heat-shock proteins IbpA and IbpB. Reasonable yield was obtained when the cells were cultured in Terrific Broth + 1% glucose medium at 22 degrees C for 16 h. The optimal cell density for expression of soluble 6xHis-ARHGAP6 protein was at A(600) about 0.5. Under these conditions over 90% of the fusion protein was present in a soluble form. The 6xHis-ARHGAP6 protein was purified to near homogeneity by a two step procedure comprising chromatography on Ni-nitrilotriacetate and cation exchange columns. The expression system and purification procedure employed made it possible to obtain 1-2 mg of pure 6xHis-ARHGAP6 protein from 300 ml (1.5 g of cells) of E. coli culture.

scholarly journals Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

2010 ◽  
Vol 192 (9) ◽  
pp. 2407-2413 ◽  
Author(s):  
Jacalyn M. Green ◽  
Ryan Hollandsworth ◽  
Lenore Pitstick ◽  
Eric L. Carter
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Size Exclusion ◽  
Breakdown Product ◽  
Purification And Characterization ◽  
Sds Page

ABSTRACT The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a Km value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min−1 mg−1. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

Cloning, Expression, Purification, and Characterization of an Organophosphate-Degrading Enzyme in Escherichia Coli

2012 ◽  
Vol 610-613 ◽  
pp. 203-209 ◽  
Author(s):  
Jin Bin Wang ◽  
Da Chao Chen ◽  
Dong Xiao Hu ◽  
Xue Feng Su ◽  
Xue Ming Tang
Keyword(s):  
Escherichia Coli ◽  
Organic Phosphorus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cloning And Expression ◽  
E Coli ◽  
Maximum Reaction ◽  
Sds Page ◽  
Protein Expression And Purification ◽  
Optimal Value

OpdA is one of organic phosphorus degradation enzyme gene from Agrobacterium radiobacter that may be the most promising targets for the digestion of digestion. In this study, we describe for the cloning and expression in Escherichia coli (E. coli) of plasmid pET28b-opdA, followed by purification by NTA-Ni2+ agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).The Results showed that the optimal value of inoculum OD600 before induction, inducing time, final IPTG concentration and inducing temperature respectively were 0.5,5h,1 mmol/L,37°C. We obtained the concentration of renatured protein was 18.312mg / L. The Km was 4.26μmol/L at 37 °C, and the maximum reaction velocity (Vmax) was 3.2669μmol/L • min.

Expression and Identification on the MPB83-MPB70 Fusion Gene of Mycobacterium bovis in Escherichia coli

2014 ◽  
Vol 997 ◽  
pp. 210-214
Author(s):  
Yun Hang Gao ◽  
Yan Liu ◽  
Ying Nan Zhang ◽  
Yan Yu ◽  
Chun Lei Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Bovis ◽  
Expression Vector ◽  
Fusion Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigenic Activity ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Single Antigen

For raising the antigenicity ofMycobacterium bovissingle antigen, fusion protein of two genes was acquired. The DNA fragments ofmpb83andmpb70were fused by splicing by overlapping extension (SOE) polymerase chain reaction (PCR), and the fusion gene mpb83-mpb70 were cloned into pMD18-T vector, then we got the recombinant plasmid pMD-83-70. pMD-83-70 and pET28a (+) were digested byBamH I andEcoR I double enzymes. The purified pMD-83-70 fusion gene was subcloned into the expression vector pET28a (+), and the prokaryotic expression vector pET-83-70 was constructed. Plasmid containing pET-83-70 was transformed into competenceEscherichia coliBL21 (DE3). The bacterium was induced by isopropyl-β-D-thiogalactoside (IPTG) and the lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 41 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed by using Western blotting, the results indicated that the protein was of antigenic activity ofM.bovis. These results could serve as a basis for further studies on the usefulness of the fusion gene and its expression product in the development of subunit vaccine and DNA vaccine against bovine tuberculosis.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

scholarly journals Chemical Modification of Nisin With Homocysteine Thiolactone And Its Antibacterial Activity

2021 ◽  
Author(s):  
Ezgi Demir Özer ◽  
Metin YILDIRIM
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Optimum Conditions ◽  
Homocysteine Thiolactone ◽  
Sds Page

Abstract The objective of this study was to prepare N-homocysteine conjugate of nisin, and to evaluate the effect of N-homocysteinylation reaction on the antibacterial activity of nisin. Progression of the modification was monitored by measuring free sulfhydryl groups, and by using both acetic acid urea polyacrylamide gel electrophoresis (AAU-PAGE) and tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (tricine SDS-PAGE). Optimum conditions to attain the maximum N-homocysteinylation degree (6.30%) were determined as 6 mg/mL nisin, 150 mM homocysteine thiolactone, 150 rpm shaker speed, 3.0 pH and 6 hrs reaction time. Antibacterial activity of N-homocysteinylated nisin was determined against Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Lactococcus lactis ssp. cremoris AÜ, Enterococcus faecium ATCC 9097, Listeria monocytogenes NCTC 5348 and Escherichia coli RSKK. Of all the bacteria tested only the growth of E. faecium was inhibited slightly by modified nisin having just about 10% of antibacterial activity of unmodified nisin. Tricine SDS-PAGE analysis applied after trypsin and thermolysin hydrolysis showed that side chains of Lys12 and Lys22 were both modified with homocysteine thiolactone reaction.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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