New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; A potential vector for a two-plasmid expression system

Plasmid ◽  
2012 ◽  
Vol 67 (3) ◽  
pp. 264-271 ◽  
Author(s):  
Piotr Zaleski ◽  
Pawel Wawrzyniak ◽  
Agnieszka Sobolewska ◽  
Diana Mikiewicz ◽  
Anna Wojtowicz-Krawiec ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression Vector ◽  
Expression System ◽  
Cloning And Expression ◽  
Potential Vector ◽  
Coli Plasmid

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

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.

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.

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.

Cloning and Expression of Taxus Acyltransferase cDNA

2004 ◽  
Vol 59 (9-10) ◽  
pp. 755-761 ◽  
Author(s):  
Jun Tu ◽  
Ping Zhu ◽  
Ke-di Cheng ◽  
Jiangguo Liu
Keyword(s):  
Escherichia Coli ◽  
Expression Vector ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Taxus Chinensis ◽  
Biosynthetic Pathways ◽  
Reading Frame ◽  
Calculated Molecular Weight ◽  
Cloning Strategy ◽  
Calculated Mass

Abstract A new full-length acyltransferase cDNA was obtained from Taxus chinensis by homologybased cloning strategy. The cDNA has an open-reading frame of 1,275 nucleotides, which encodes 425 amino acids with a calculated molecular weight of 47,241 Da and an estimated pI value of 5.93. The deduced amino acid sequence resembles the sequences of other cloned acyltransferases (56-61% identity; 71-75% similarity) involved directly in taxol biosynthetic pathways. This cDNA was expressed in Escherichia coli using the expression vector pET- 32a(+). The expression band corresponds to the calculated mass plus the N-terminal fusion protein derived from the vector.

Cloning and Expression of Antibacterial Goat Lactoferricin from Escherichia coli AD494(DE3)pLysS Expression System

2008 ◽  
Vol 71 (12) ◽  
pp. 2523-2525 ◽  
Author(s):  
GEN-HUNG CHEN ◽  
LI-JUNG YIN ◽  
I-HUA CHIANG ◽  
SHANN-TZONG JIANG
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Propionibacterium Acnes ◽  
Expression System ◽  
Cloning And Expression ◽  
Soluble Form ◽  
Diffusion Method ◽  
Activity Assay ◽  
E Coli ◽  
Expression Host

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-β-d -thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His·Tag and GLfcin S-His·Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His·Tag and GLfcin S-His·Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His·Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His·Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100°C.

scholarly journals Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System

2016 ◽  
Vol 6 (2) ◽  
pp. 187-194 ◽  
Author(s):  
Abbas Mohajeri ◽  
Yones Pilehvar-Soltanahmadi ◽  
Mohammad Pourhassan-Moghaddam ◽  
Jalal Abdolalizadeh ◽  
Pouran Karimi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Cloning And Expression ◽  
Recombinant Human Endostatin ◽  
Escherichia Coli Expression
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