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 Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export

2021 ◽  
Vol 12 ◽  
Author(s):  
Jakob H. Viel ◽  
Amanda Y. van Tilburg ◽  
Oscar P. Kuipers
Keyword(s):  
Antimicrobial Activity ◽  
Synthetic Biology ◽  
Heterologous Expression ◽  
Expression System ◽  
Heterologous Expression System ◽  
Proteolytic Activation ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Precursor Peptide ◽  
Potential Applications

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.

Cloning and expression of l-asparaginase from E. coli in eukaryotic expression system

2015 ◽  
Vol 102 ◽  
pp. 14-17 ◽  
Author(s):  
Syed Sajitha ◽  
Jalaja Vidya ◽  
karunakaran Varsha ◽  
Parameswaran Binod ◽  
Ashok Pandey
Keyword(s):  
Expression System ◽  
Eukaryotic Expression ◽  
Cloning And Expression ◽  
E Coli ◽  
Eukaryotic Expression System

scholarly journals Bone morphogenetic protein 2: heterologous expression and potential in bone regeneration

2021 ◽  
Vol 43 (114) ◽  
Author(s):  
Andrea Mesa Restrepo ◽  
Juan Fernando Alzate ◽  
Edwin Bairon Patiño Gonzalez
Keyword(s):  
Heterologous Expression ◽  
Bone Morphogenetic Protein ◽  
Recombinant Dna ◽  
Redox System ◽  
Bone Morphogenetic Protein 2 ◽  
Recombinant Dna Technology ◽  
Morphogenetic Protein ◽  
Alkaline Phosphate ◽  
Heterologous Expression Systems

Currently, bone morphogenetic protein 2 (BMP-2) is one of the two osteoinductive growth factors used in medical devices to promote bone formation. Typically, this protein is bought from commercial houses at high rates and in small quantities that are not enough to cover clinical needs. Because of this, it has been proposed that research centers use their own heterologous expression systems to have a constant supply of BMP-2. The aim of this study was to standardize the heterologous expression of BMP-2 and evaluate its osteoinductive activity in vitro. Our procedure for expression and purification was based on recombinant DNA technology using the plasmid pET-28 and IPTG as inductor. After extracting the protein from inclusion bodies, folding it and modifying it via a redox system, we observed via electrophoresis a 26 kDa dimer. We evaluated its osteoinductive activity in myoblastic C2C12 by quantifying enzymatically the activity of alkaline phosphate (ALP) and staining mineralization nodules. ALP activity is proportional to BMP-2 concentration, increasing 90% at 3 µg/mL. These cells form calcium nodules, mineralizing 50% of the area.

Biological Production of Uniform Polypeptides for Optical Applications

1989 ◽  
Vol 174 ◽  
Author(s):  
Carl W. Lawton ◽  
Herbert E. Klei ◽  
Linda D. Strausbaugh ◽  
Robert Crane
Keyword(s):  
Recombinant Dna ◽  
Narrow Band ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Recombinant Dna Technology ◽  
Base Pairs ◽  
Synthetic Genes ◽  
Optical Applications ◽  
Nonlinear Optical Applications

AbstractRecent advances in recombinant DNA technology have created the potential for engineering of protein molecules to specific uses beyond those normally considered for biomaterials. This research project has demonstrated the feasibility of producing polypeptides useful for narrow band filters and nonlinear optical applications.Synthetic genes, ranging in size from 36 to 576 base pairs, have been constructed from oligonucleotides using a restriction doubling technique. The synthetic genes have been inserted into a Protein A fusion expression system. Fused polypeptides from induced cells have been purified by affinity chromatography (IGG), and analyzed by polyacrylamide gel electrophoresis.

Synthesis of a Collagen Analog in Escherichia Coli Using Recombinant DNA Technology

1989 ◽  
Vol 174 ◽  
Author(s):  
Ina Goldberg ◽  
A. J. Salerno
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Tandem Repeats ◽  
Recombinant Dna ◽  
Cellular Proteins ◽  
Recombinant Dna Technology ◽  
Synthetic Genes ◽  
E Coli ◽  
Bacterial Protease

AbstractA family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the Clal cloning site of the expression vector pJL6. A representative recombinant plasmid, pACI, with an insert of about 340 bp, was established in an Escherichia coli strain bearing a defective λ prophage, to study expression of the CII-collagen analog fusion protein produced from pACI upon heat induction. The in vivo levels of synthetic gene expression obtained showed that the fusion protein was synthesized in E. coli, but was labile compared to other cellular proteins. This degradation could be significantly reduced by the genetic inhibition of a bacterial protease system.

Recombination in yeast and the recombinant DNA technology

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 536-540 ◽  
Author(s):  
Thomas D. Petes ◽  
Peter Detloff ◽  
Sue Jinks-Robertson ◽  
S. Renee Judd ◽  
Martin Kupiec ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Recombinant Dna ◽  
Recombinant Dna Technology ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Genes ◽  
Dna Technology ◽  
Recombinant Dna Techniques ◽  
Repeated Genes

The development of methods to isolate eukaryotic genes, alter these genes in vitro and reintroduce them into the cell has had a major impact on the study of recombination in the yeast Saccharomyces cerevisiae. In this paper we discuss how recombinant DNA techniques have been employed in the study of recombination in yeast and the results that have been obtained in these studies.Key words: recombination, Saccharomyces cerevisiae, gene conversion, repeated genes.

scholarly journals Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication

2001 ◽  
Vol 82 (7) ◽  
pp. 1767-1776 ◽  
Author(s):  
Jianhe Huang ◽  
David B. Levin
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Spodoptera Littoralis ◽  
Expression System ◽  
Functional Characterization ◽  
Eukaryotic Expression ◽  
Replication Assay ◽  
E Coli ◽  
Origin Of Dna Replication

The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3′–5′ exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.

scholarly journals Escherichia coli BL21(DE3) expression system using TorA signal peptide for Recombinant Human Albumin (rHA) secretion

2019 ◽  
Vol 10 (4) ◽  
pp. 3319-3324 ◽  
Author(s):  
Iman P. Maksum ◽  
Astri Lestari ◽  
Retna P. Fauzia ◽  
Saadah D. Rachman ◽  
Ukun M.S. Soedjanaatmadja
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Signal Peptide ◽  
Recombinant Dna ◽  
Expression System ◽  
Foreign Protein ◽  
Competent Cell ◽  
Promising Alternative ◽  
E Coli ◽  
Sds Page

Human serum albumin (HSA) is the most abundant protein in blood plasm. This protein consisted of 585 amino acids with a molecular weight of 66 kDa and 17 disulfide bonds. HSA obtained from conventional technique allow viral or prion contamination. For that reason, recombinant DNA technology becomes a promising alternative. Because of its well-known genetic, simplicity, and capacity to accommodate many foreign protein, Escherichia coli remains the most widely used in the production of recombinant proteins. But, overproduction of protein may lead to the formation of inclusion bodies and proteolytic degradation. These problems can be overcome by using protease-deficient strain and protein secretion into periplasmic space. The objective of this research is to secrete recombinant HA on E. coli BL21(DE3) using TorA signal peptide and proved using SDS-PAGE. This research method begins with the preparation of competent cell and transformation of E. coli BL21(DE3), expression of recombinant HA in E. coli BL21(DE3), and characterization of expression result by using SDS-PAGE. The result of this study was rHSA can be secreted into extracellular medium using TorA signal peptide with a molecular weight of ± 66.5 kDa.

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