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

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.

The Quiescent-Cell Expression System for Protein Synthesis in Escherichia coli

1999 ◽  
Vol 65 (6) ◽  
pp. 2710-2715 ◽  
Author(s):  
Duncan C. D. Rowe ◽  
David K. Summers
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Total Cell ◽  
Quiescent Cell ◽  
Cell Protein ◽  
Quiescent Cells ◽  
Protein Overproduction ◽  
Mutant Strains ◽  
Cell Expression ◽  
Cell Expression System

ABSTRACT The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction inEscherichia coli. It is dependent on the controlled overexpression of a small RNA called Rcd in hns mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.

Expression of Functional Recombinant Mussel Adhesive Protein Type 3A in Escherichia coli

2008 ◽  
Vol 21 (3) ◽  
pp. 965-970 ◽  
Author(s):  
Dong Soo Hwang ◽  
Youngsoo Gim ◽  
Hyung Joon Cha
Keyword(s):  
Escherichia Coli ◽  
Adhesive Protein ◽  
Mussel Adhesive Protein ◽  
Mussel Adhesive

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 Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

2013 ◽  
Vol 16 (1) ◽  
pp. 13-22 ◽  
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.

scholarly journals Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-->Gln and Glu73-->Gln mutants.

1997 ◽  
Vol 44 (2) ◽  
pp. 275-283 ◽  
Author(s):  
K Bolewska ◽  
H Kozłowska ◽  
G Goch ◽  
B Mikołajek ◽  
A Bierzyński
Keyword(s):  
Escherichia Coli ◽  
Calcium Binding ◽  
Expression System ◽  
Total Yield ◽  
Site Directed Mutagenesis ◽  
Cloning And Expression ◽  
Alpha Subunits ◽  
C Terminus ◽  
Gene Coding ◽  
Native Sequence

Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.

Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli

1989 ◽  
Vol 3 (1) ◽  
pp. 15-21 ◽  
Author(s):  
M. C. Hanks ◽  
R. T. Talbot ◽  
H. M. Sang
Keyword(s):  
Escherichia Coli ◽  
Cdna Sequence ◽  
Expression Vector ◽  
Total Cell ◽  
Biologically Active ◽  
Cell Protein ◽  
Western Blots ◽  
E Coli ◽  
Ring Dove

ABSTRACT The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK233-2 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1·5% of total cell protein.

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