Cloning and expression of the insecticidal toxin gene “tccB” from Photorhabdus temperata M1021 in Escherichia coli expression system

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.

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Expression of adenylate kinase fused mouse ubiquitin active enzyme in Escherichia coli and its application in ubiquitination

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab026 ◽

2021 ◽

Author(s):

Xiaoliang Liu ◽

Ling Hu ◽

Yuan Zhang ◽

Hongtao Li

Keyword(s):

Escherichia Coli ◽

Catalytic Activity ◽

Chemical Synthesis ◽

Adenylate Kinase ◽

Energy Supply ◽

Expression System ◽

Synthesis Process ◽

Cellular Functions ◽

Escherichia Coli Expression

Abstract Ubiquitination, is involved in the regulation of numerous cellular functions. Researches in the ubiquitin realm rely heavily on ubiquitination assays in vitro and require large amounts of ubiquitin-activating enzyme (UBA1) and keep ATP supplies. But UBA1 is hard to be obtained with large quantities using reported methods. We fused Escherichia coli adenylate kinase (adk) and mouse UBA1 obtained fusion protein adk-mUBA1. The expression level of adk-mUBA1 increased about 8-fold than that of mUBA1 in Escherichia coli expression system, and adk-mUBA1 was easily purified to 90% purity via two purification steps. The purified adk-mUBA1 protein was functional for ubiquitination and could use ATP in addition to ADP as energy supply and had a higher catalytic activity than mUBA1 in cell lysis. Adk-mUBA1 can be applied to preparing ubiquitin modified substrates and kinds of ubiquitin chains in chemical synthesis process and is preferable application than mUBA1 in vitro ubiquitination.

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Cotranslational Incorporation of a Structurally Diverse Series of Proline Analogues in an Escherichia coli Expression System

ChemBioChem ◽

10.1002/cbic.200400052 ◽

2004 ◽

Vol 5 (7) ◽

pp. 928-936 ◽

Cited By ~ 49

Author(s):

Wookhyun Kim ◽

Anna George ◽

Melissa Evans ◽

Vincent P. Conticello

Keyword(s):

Escherichia Coli ◽

Expression System ◽

Escherichia Coli Expression

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Effect of rhBMP-2 produced by Escherichia coli expression system on bone formation in rat calvarial defects

The Journal of the Korean Academy of Periodontology ◽

10.5051/jkape.2009.39.1.77 ◽

2009 ◽

Vol 39 (1) ◽

pp. 77

Author(s):

Suk-Hoon Kwon ◽

Hyun-Chang Lim ◽

Kyung-Hee Choi ◽

Min-Soo Kim ◽

Ji-Hyun Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Bone Formation ◽

Expression System ◽

Calvarial Defects ◽

Escherichia Coli Expression

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Cloning and Expression of a Synthetic Mussel Adhesive Protein in Escherichia Coli

MRS Proceedings ◽

10.1557/proc-292-99 ◽

1992 ◽

Vol 292 ◽

Author(s):

Anthony J. Salerno ◽

Ina Goldberg

Keyword(s):

Escherichia Coli ◽

Soluble Fraction ◽

Expression System ◽

Total Cell ◽

Cloning And Expression ◽

Vector System ◽

Cell Protein ◽

Adhesive Protein ◽

Mussel Adhesive ◽

Unit Repeat

AbstractRepetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were cloned and expressed in Escherichia coli. The bioadhesive precursor (BP, Mx−25,000) was expressed from one 600-bp gene comprised of a 30-bp unit repeat. The repetitious gene appeared stable in a T7-based host/vector system.Using the T7 expression system for induction, BP was produced at levels approaching 60% of total cell protein. BP was found both in intracellular inclusions and in the soluble fraction. Interestingly, methionine was processed from the N-terminus of the purified protein to give an authentic consensus precursor protein.

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Production of an authentic-like recombinant protein based on Escherichia coli expression system

Seibutsu Butsuri ◽

10.2142/biophys.40.s185_4 ◽

2000 ◽

Vol 40 (supplement) ◽

pp. S185

Author(s):

T. Koshiba ◽

T. Tamada ◽

K. Nitta ◽

R. Kuroki

Keyword(s):

Escherichia Coli ◽

Recombinant Protein ◽

Expression System ◽

Escherichia Coli Expression

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Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

Science and Technology Development Journal ◽

10.32508/stdj.v16i1.1392 ◽

2013 ◽

Vol 16 (1) ◽

pp. 13-22 ◽

Cited By ~ 1

Author(s):

Trang Thi Phuong Phan ◽

Anh Le Tuan Nguyen ◽

Hoang Duc Nguyen

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Fusion Protein ◽

Expression System ◽

Pharmaceutical Products ◽

Cloning And Expression ◽

Gene Encoding ◽

E Coli ◽

B Subunit ◽

The Common

LTB is the B subunit of heat labile toxins (LT) in Escherichia coli ETEC. This subunit is non-toxic but has a high immune response. Therefore, LTB is considered a suitable antigen for partial vaccine against the diarrhea caused by E. coli ETEC. The most important component of partial vaccine is antigen protein. Nowadays, with the advancement of recombinant protein technology, these antigens are mainly produced by the common bacterial expression system as E. coli. However, the recombinant proteins produced by E. coli are often miscellaneous with enterotoxins, which should be removed from pharmaceutical products. Thus, the production of antigen proteins in other expression system without endotoxins like Bacillus subtilis is in attention. We conducted the experiments of cloning and expressing LTB using a novel pHT plasmid that allow the protein to be expressed in both of E. coli and B. subtilis. We were successful to generate plasmid pHT326 and express the gene encoding for the fusion protein of LTB and LysSN-6xHis-TEV in B. subtilis and E. coli. The binding of fusion protein on the columns that have affinity with His-tag was confirmed. This result is about to be applied for the development of partial vaccine aganst the diarrhea as well as the development of some diagnostic kits for ETEC in food or medical waste and kits to detect antibodies against LTB in animals.

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