A semi-continuous process for the production of human interferon-?c from E. coli using tangential-flow microfiltration and immuno-affinity chromatography

1984 ◽  
Vol 6 (10) ◽  
pp. 621-626 ◽  
Author(s):  
B. Vaks ◽  
Y. Mory ◽  
J. U. Pederson ◽  
O. Horovitz
Keyword(s):  
Affinity Chromatography ◽  
Continuous Process ◽  
Human Interferon ◽  
E Coli ◽  
Tangential Flow

scholarly journals Production of Human Interferon β by Recombinant E. coli Using the Codon Optimized Gene

KSBB Journal ◽  
2017 ◽  
Vol 32 (1) ◽  
pp. 16-21
Author(s):  
Jong-Seok Kim ◽  
Seung-Won Jang ◽  
Jae-Bum Park ◽  
Deok-Ho Kwon ◽  
Young-Jun Chang ◽  
...  
Keyword(s):  
Human Interferon ◽  
Interferon Β ◽  
E Coli

scholarly journals Heterologous Expression and Purification of ? Galactosidase Protein Using Affinity Chromatography

2013 ◽  
Vol 5 (3) ◽  
pp. 499-513
Author(s):  
M. Z. Alam ◽  
L. Ragionieri ◽  
M. A. S. Santos ◽  
A. Iqbal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Affinity Chromatography ◽  
Recombinant Dna ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Lacz Gene ◽  
Recombinant Dna Technology ◽  
E Coli ◽  
Elution Buffer

Enzymes and other protein purification using recombinant DNA technology have become popular due to scarcity of natural protein. Saccharomyces cerevisiae is a demanding host, since it facilitates protein expression by its relative simplicity, safe organisms, inexpensive and has many properties of eukaryotic expression system. As an alternative host we express E. coli lacZ gene with GST tag in Saccharomyces cerevisiae and successfully purified from soluble extracts. The concentration of soluble GST-? galactosidase protein was approximately 0.57 mg/ml of elution buffer yielded from 50 ml yeast cell culture. The ?-galactosidase protein from insoluble extract was low due to the increasing solubility of GST tag. Keywords: ?-galactosidase; Heterologous expression; GST tag; Affinity chromatography. © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v5i3.13820 J. Sci. Res. 5 (3), 499-513 (2013)  

scholarly journals pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

2001 ◽  
Vol 67 (3) ◽  
pp. 1262-1267 ◽  
Author(s):  
Shuhei Fujimoto ◽  
Yasuyoshi Ike
Keyword(s):  
Affinity Chromatography ◽  
Protein Purification ◽  
Enterococcus Faecalis ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Chromatography Method ◽  
Shuttle Vectors ◽  
E Coli ◽  
One Step

ABSTRACT Two novel Enterococcus faecalis-Escherichia colishuttle vectors that utilize the promoter and ribosome binding site ofbacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli andE. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coliand E. faecalis at levels of 103 and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.

scholarly journals Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

2022 ◽  
Vol 82 ◽  
Author(s):  
Fernanda Ghenov ◽  
Edileusa Cristina Marques Gerhardt ◽  
Luciano Fernandes Huergo ◽  
Fabio Oliveira Pedrosa ◽  
Roseli Wassem ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Glutamine Synthetase ◽  
Affinity Chromatography ◽  
Azospirillum Brasilense ◽  
Nitrogen Assimilation ◽  
Atp Hydrolysis ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
E Coli

Abstract Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

scholarly journals Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System

Data ◽  
2021 ◽  
Vol 6 (9) ◽  
pp. 99
Author(s):  
Rafida Razali ◽  
Vijay Kumar Subbiah ◽  
Cahyo Budiman
Keyword(s):  
Affinity Chromatography ◽  
Drug Targets ◽  
Single Step ◽  
Host Cells ◽  
Soluble Form ◽  
Expression And Purification ◽  
Technical Data ◽  
E Coli ◽  
Main Protease ◽  
Chromatography Step

The SARS-CoV-2 coronavirus expresses two essential proteases: firstly, the 3Chymotrypsin-like protease (3CLpro) or main protease (Mpro), and secondly, the papain-like protease (PLpro), both of which are considered as viable drug targets for the inhibition of viral replication. In order to perform drug discovery assays for SARS-CoV-2, it is imperative that efficient methods are established for the production and purification of 3CLpro and PLpro of SARS-CoV-2, designated as 3CLpro-CoV2 and PLpro-CoV2, respectively. This article expands the data collected in the attempts to express SARS-CoV-2 proteases under different conditions and purify them under single-step chromatography. Data showed that the use of E. coli BL21(DE3) strain was sufficient to express 3CLpro-CoV2 in a fully soluble form. Nevertheless, the single affinity chromatography step was only applicable for 3CLpro-CoV2 expressed at 18 °C, with a yield and purification fold of 92% and 49, respectively. Meanwhile, PLpro-CoV2 was successfully expressed in a fully soluble form in either BL21(DE3) or BL21-CodonPlus(DE3) strains. In contrast, the single affinity chromatography step was only applicable for PLpro-CoV2 expressed using E. coli BL21-CodonPlus(DE3) at 18 or 37 °C, with a yield and purification fold of 86% (18 °C) or 83.36% (37 °C) and 112 (18 °C) or 71 (37 °C), respectively. The findings provide a guide for optimizing the production of SARS-CoV-2 proteases of E. coli host cells.

scholarly journals Clonning and expression of recombinant carbapenemase KPC-2 enzyme in E. coli cytoplasm

2016 ◽  
Vol 19 (2) ◽  
pp. 27-37
Author(s):  
Phuong Nhat Tran ◽  
Phuong Thi Kim Huynh ◽  
Trang Thi Phuong Tran ◽  
Thuoc Linh Tran ◽  
Van Hung Pham
Keyword(s):  
Klebsiella Pneumoniae ◽  
Clinical Research ◽  
Affinity Chromatography ◽  
Recombinant Plasmid ◽  
Broth Culture ◽  
E Coli ◽  
Recombinant Vector ◽  
Carbapenem Resistant ◽  
Sds Page ◽  
Encoding Genes

Production of KPC-type carbapenemase is the most common carbapenem resistant mechanism in Klebsiella pneumoniae. The expression level of KPC in these strains is different and is mostly required other mechanisms to reach the higher resistant level such as porin lost or co-expression of extended spectrum β-lactamase (ESBL). To better understand the expression of KPC enzyme, the KPC-2 encoding genes from clinical isolated K. pneumoniae were cloned into pET28a plasmid. The recombinant plasmids containing of kpc-2 gene were subsequently transformed into E. coli OmniMax and were screened in kanamycine added LB media to select E. coli possessing of recombinant plasmid. Carbapenemase activity in the broth culture was checked in LB broth supplemented with 4 µg/mL of ertapenem and the expression induced with IPTG was checked by SDS-PAGE method. The results showed that this recombinant vector was capable of effective expression of KPC-2 protein in E. coli and this strain could be grown in LB broth supplemented with 4 µg/mL of ertapenem. A half of the target protein was soluble in the supernatant however it could be successfully collected from a HistrapHP affinity chromatography column. The result of this report is one of resources for further studies and applications of this KPC-2 protein in clinical research.

scholarly journals Identification and Characterization of a Novel Ibe10 Binding Protein That Contributes to Escherichia coliInvasion of Brain Microvascular Endothelial Cells

1999 ◽  
Vol 67 (3) ◽  
pp. 1131-1138 ◽  
Author(s):  
Nemani V. Prasadarao ◽  
Carol A. Wass ◽  
Sheng-He Huang ◽  
Kwang Sik Kim
Keyword(s):  
Endothelial Cells ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Affinity Chromatography ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
E Coli ◽  
Blood Brain

ABSTRACT The molecular basis of Escherichia coli traversal of the blood-brain barrier in the development of E. colimeningitis is not well understood. We have previously shown that a novel Ibe10 protein found in cerebrospinal fluid isolates of E. coli is necessary for invasion of the brain microvascular endothelial cells (BMEC) that constitute the blood-brain barrier both in vitro and in a newborn rat model of hematogenous meningitis. Here we identified a novel Ibe10 binding molecule/receptor (Ibe10R) on both bovine BMEC (HBMEC) and human BMEC (HBMEC) that is responsible for invasion by E. coli. Ibe10R, an approximately 55-kDa protein, was purified from BBMEC by Ibe10-Ni-Sepharose affinity chromatography. Bovine Ibe10R, as well as polyclonal antibodies to Ibe10R, blocked E. coli invasion of BBMEC very effectively. The N-terminal amino acid sequence of Ibe10R showed 75% homology to serum albumin. However, the amino acid sequence of an Ibe10R fragment generated by limited enzymatic digestion did not reveal homology to any other proteins, suggesting that Ibe10R represents a novel albumin-like protein. Immunocytochemical analysis of BBMEC using anti-Ibe10R antibody suggested that only a subset of cultured BBMEC express Ibe10R on their surface. Enrichment of Ibe10R-positive BBMEC by fluorescence-activated cell sorting with anti-Ibe10R antibody resulted in enhanced invasion by E. coli. The anti-Ibe10R antibody raised against bovine Ibe10R also blocked E. coli invasion of HBMEC very effectively. Interestingly, anti-Ibe10R antibody affinity chromatography of HBMEC membrane proteins revealed a smaller protein with an approximate molecular mass of 45 kDa. These results suggest that the Ibe10 of E. coli interacts with a novel BMEC surface protein, Ibe10R, for invasion of both BBMEC and HBMEC.

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