scholarly journals The Effect of Single Co-expression of The DnaK-DnaJ-GrpE and GroEL/ES Chaperones and Their Combination on Expression Intein-pretrombin-2 in Escherichia coli ER2566

2020 ◽  
Vol 6 (1) ◽  
pp. 47-54
Author(s):  
Iman Permana Maksum ◽  
D Agus Yusuf Wildan ◽  
Khomaini Hasan ◽  
Toto Subroto
Keyword(s):  
Inclusion Bodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iptg Induction ◽  
E Coli ◽  
Expression Host ◽  
Sds Page ◽  
Culture Growth ◽  
The One ◽  
Cell Culture Growth

The use of recombinant thrombin in the manufacture of fibrin glue allows diseases contamination to be avoided. However, the expression of recombinant protein in E. coli still has a disadvantage of the formation of inclusion bodies, so it needs to be minimized by co-expression of chaperones. Therefore, the aim of this study was to determine the effect of single DnaK-DnaJ-GrpE and GroEL/ES chaperone expression and their combination on the expression of intein-pretrombin-2Ti,pH on E. coli ER2566. The method started with isolation of pTWIN1-prethrombin-2Ti,pH and pG-KJE8 from E. coli TOP10F' and DH5α respectively, the co-transformation of the expression host E. coli ER2566 using pG-KJE8 and pTWIN1-prethrombin-2Ti,pHvectors, the chaperone co-expression was induced using L-Arabinosa before IPTG induction and cell culture growth was incubated at 22 oC. The expression products were characterized by using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results of the co-expression of chaperone showed that the number of soluble fraction was higher than the one without co-expression of chaperone. In addition, the co-expression of chaperone using pG-KJE8 in intein-prothrombin-2Ti,pH expression was sufficient using tetracycline as an inducer.

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.

scholarly journals Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Fatemah Bakhshi ◽  
Reza Pilehchian Langroudi ◽  
Bahram Golestani Imani
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Clostridium Perfringens ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Toxin Gene ◽  
Type B ◽  
Iptg Induction ◽  
E Coli ◽  
Beta Toxin

Clostridium perfringens beta toxin is only produced by types B and C and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. We compared the expression of C. perfringens type B vaccine strain recombinant beta toxin gene in the Escherichia coli strains RosettaTM(DE3) and BL21(DE3). The beta toxin gene was extracted from pJETβ and ligated with pET22b(+). pET22β was transformed into E. coli strains BL21(DE3) and RosettaTM(DE3). Recombinant protein was expressed as a soluble protein after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction in strain RosettaTM(DE3) but not in BL21(DE3). Expression was optimised by growing recombinant cells at 37 °C and at an induction of 0.5 mM, 1 mM, 1.5 mM IPTG. Expression was evaluated using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The recombinant protein was purified via Ni-NTA and was analysed using western blot. We concluded that E. coli strain RosettaTM(DE3) can enhance the expression of C. perfringens recombinant beta toxin.Keywords: C. perfringens beta toxin (CPB); expression; RosettaTM; BL21

scholarly journals Immunochemical studies on R mutants of Yersinia enterocolitica O:3.

1998 ◽  
Vol 45 (4) ◽  
pp. 1011-1019 ◽  
Author(s):  
J Radziejewska-Lebrecht ◽  
M Skurnik ◽  
A S Shashkov ◽  
L Brade ◽  
A Rózalski ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Inner Core ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nmr Studies ◽  
E Coli ◽  
Enterobacterial Common Antigen ◽  
Sds Page ◽  
K 12 ◽  
Specific Sugar

Three mutants of Yersinia enterocolitica O:3, namely: YeO3-R1, YeO3-RfbR7 and YeO3-c-trs8-R were classified on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) profile of isolated lipopolysaccharides (LPS) as belonging to the Ra- (the first) and the Rc-type (the other two mutants). Methylation analysis, in addition to 13C and 1H NMR studies of purified core oligosaccharides revealed structures similar to those established previously for the full core of Y. enterocolitica O:3 in the case of the Ra mutant, and identical to that reported for the Rc mutant Ye75R, in the case of the two other mutants. The O-specific sugar, 6d-L-altrose, which forms a homopolymeric O-chain, was present in small amounts in all three LPS preparations, as well as in the core oligosaccha ride preparations along with the Ra and the Rc sugars, characteristic of the Y. enterocolitica O:3 core. This result is in line with genetic data, indicating that it is the inner core region which is the receptor for the O-specific chain in Y. enterocolitica O:3. This region seems likewise to be the anchoring region for the enterobacterial common antigen (ECA), as shown by SDS/PAGE/Western blot analysis with monoclonal antibodies against ECA. In addition, we also demonstrated that the Ye75R mutant Rc and its parental strain Ye75S, both were ECA-immunogenic strains. So far, ECA-immunogenic strains, i.e. those with LPS-linked ECA, were only identified in E. coli mutants of the R1, R4 and K-12 serotype.

scholarly journals Interactions of an Essential Bacillus subtilis GTPase, YsxC, with Ribosomes

2007 ◽  
Vol 190 (2) ◽  
pp. 681-690 ◽  
Author(s):  
Catherine Wicker-Planquart ◽  
Anne-Emmanuelle Foucher ◽  
Mathilde Louwagie ◽  
Robert A. Britton ◽  
Jean-Michel Jault
Keyword(s):  
Bacillus Subtilis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Small Gtpase ◽  
E Coli ◽  
Protein Partners ◽  
The One

ABSTRACT YsxC is a small GTPase of Bacillus subtilis with essential but still unknown function, although recent works have suggested that it might be involved in ribosome biogenesis. Here, purified YsxC overexpressed in Escherichia coli was found to be partly associated with high-molecular-weight material, most likely rRNA, and thus eluted from gel filtration as a large complex. In addition, purification of ribosomes from an E. coli strain overexpressing YsxC allowed the copurification of the YsxC protein. Purified YsxC was shown to bind preferentially to the 50S subunit of B. subtilis ribosomes; this interaction was modulated by nucleotides and was stronger in the presence of a nonhydrolyzable GTP analogue than with GTP. Far-Western blotting analysis performed with His6-YsxC and ribosomal proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that YsxC interacted with at least four ribosomal proteins from the 50S subunit. Two of these putative protein partners were identified by mass spectrometry as L1 and L3, while the third reactive band in the one-dimensional gel contained L6 and L10. The fourth band that reacted with YsxC contained a mixture of three proteins, L7/L12, L23, and L27, suggesting that at least one of them binds to YsxC. Coimmobilization assays confirmed that L1, L6, and L7/L12 interact with YsxC. Together, these results suggest that YsxC plays a role in ribosome assembly.

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.

Effect of Sericin Concentration on the Growth and Morphology of Escherichi Coli

2015 ◽  
Vol 815 ◽  
pp. 451-457 ◽  
Author(s):  
Rui Xue ◽  
Bing Jie Chen ◽  
Xiao Jin ◽  
Qing Song Zhang ◽  
Mei Ling Han ◽  
...  
Keyword(s):  
Cell Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Growth Curves ◽  
E Coli ◽  
Carbon And Nitrogen ◽  
Sds Page ◽  
Great Possibility ◽  
Functional Biomaterials ◽  
Escherichi Coli

Silk sericin composed of 18 amino acids has been widely used in the fields of cosmetic additives, food, medicine and functional biomaterials because of good hydrophilicity and biocompatibility, making it great possibility in providing abundant nutrients for microbial growth. Sericin (40~200 KDa) was used as culture medium for incubation of E. coli at 37°C to study the effect of sericin concentration on the growth of bacterial Escherichi coli (E. coli). The growth curves of E. coli, surface/inside morphology and protein of E. coli were investigated by UV/vis spectrophotometer (UV/vis), scanning electron microscopy (SEM), transmission electronic microscopy (TEM) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cytotoxicity of sercin was also confirmed by MTT assay. The value of OD600 increases with increasing sericin concentration from 0 to 40 g/L. Compared with the control, OD600 of 40 g/L sericin medium increases from 0.013 to 1.269 after incubated 12h. E. coli cell still remains rod shape regardless of concentration of sericin. The content of cellular soluble proteins significantly increases in sericin-treated bacteria, which in turn influenced the cell structure composition and catalyzing activity of enzyme, and finally stimulated the proliferation of E. coli. Results indicate that sericin can independently provide carbon and nitrogen for bacterial growth. Besides, it can promote bacterial protein expression without affecting cell morphology.

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 Expression and protease characterization of a conserved protein YgjD in Vibrio harveyi

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9061
Author(s):  
Yayuan Zhang ◽  
Jixiang Chen ◽  
Yonggang Wang ◽  
Yanlin Li ◽  
Wenhong Rui ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ethyl Ester ◽  
Protease Activity ◽  
Vibrio Harveyi ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bacteria Growth ◽  
E Coli ◽  
Sds Page

The glycopeptidase GCP and its homologue proteins are conserved and essential for survival of bacteria. The ygjD gene (Glycopeptidase homologue) was cloned from Vibrio harveyi strain SF-1. The gene consisted of 1,017 bp, which encodes a 338 amino acid polypeptide. The nucleotide sequence similarity of the ygjD gene with that of V. harveyi FDAARGOS 107 was 95%. The ygjD gene also showed similarities of 68%, 67% and 50% with those of Salmonella enterica, Escherichia coli and Bacillus cereus. The ygjD gene was expressed in E. coli BL21 (DE3) and the recombinant YgjD was purified by Ni2+ affinity chromatography column. The purified YgjD showed a specific 37 kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and exhibited protease activities of 59,000 units/mg, 53,700 units/mg and 8,100 units/mg, respectively, on N-Acetyl-L-tyrosine ethyl ester monohydrate (ATEE), N-Benzoyl-L-tyrosine ethyl ester (BTEE) and N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) substrates. When the conserved amino acids of His111, Glu113 and His115 in the YgjD were replaced with alanine, respectively, the protease activities of the mutants were partly decreased. The two conserved His111 and His115 of YgjD were mutated and the protein lost the protease activity, which implied that the two amino acid played very important roles in maintaining its protease activity. The addition of the purified YgjD to the culture medium of V. harveyi strain SF-1 can effectively promote the bacteria growth. These results indicated that the protease activities may be involved in the survival of bacteria.

scholarly journals MOLECULAR EXPRESSION OF WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY MEMBER 4 GENE USING Escherichia coli BL21

2017 ◽  
Vol 11 (1) ◽  
pp. 11-14
Author(s):  
Agung Janika Sitasiwi ◽  
Wayan Tunas Artama ◽  
Agung Budiyanto ◽  
Edy Dharmana
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Dna ◽  
Integration Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Negative Control ◽  
Luria Bertani ◽  
Iptg Induction ◽  
E Coli

This research was conducted to find out the Wnt4 recombinant proteins which expressed by Escherichia coli (E. coli) BL21 carrying the recombinant DNA wnt4 (E. coli transformation). Research materials were E. coli BL21 transformation and E. coli BL21 non-transformation (negative control). The expression of recombinant protein was conducted by culturing E. coli for 24 hours in Luria-Bertani (LB) media with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Recombinant protein was isolated by sonication of pellet bacteria. Protein analysis performed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that recombinant protein with a molecular weight of 33 kDa has been expressed by E. coli BL21 transformation successfully. 

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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