scholarly journals Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Md. Fakruddin ◽  
Reaz Mohammad Mazumdar ◽  
Khanjada Shahnewaj Bin Mannan ◽  
Abhijit Chowdhury ◽  
Md. Nur Hossain
Keyword(s):  
Mass Production ◽  
Recombinant Dna ◽  
Critical Factors ◽  
Molecular Tools ◽  
Recombinant Enzymes ◽  
Recombinant Dna Technology ◽  
Factors Affecting ◽  
E Coli ◽  
Industrial Level ◽  
Protein Enzyme

E. coli is the most frequently used host for production of enzymes and other proteins by recombinant DNA technology. E. coli is preferable for its relative simplicity, inexpensive and fast high-density cultivation, well-known genetics, and large number of compatible molecular tools available. Despite all these advantages, expression and production of recombinant enzymes are not always successful and often result in insoluble and nonfunctional proteins. There are many factors that affect the success of cloning, expression, and mass production of enzymes by recombinant E. coli. In this paper, these critical factors and approaches to overcome these obstacles are summarized focusing controlled expression of target protein/enzyme in an unmodified form at industrial level.

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)  

Enzymatic Research Having Pharmaceutical Significance

2018 ◽  
pp. 141-158
Author(s):  
Ishan H. Raval ◽  
Arvind Kumar Singh Chandel
Keyword(s):  
Activation Energy ◽  
Pharmaceutical Industry ◽  
Molecular Biology ◽  
Recombinant Dna ◽  
Recombinant Enzymes ◽  
Recombinant Dna Technology ◽  
Biochemical Investigation ◽  
Treatment Information ◽  
Dna Technology ◽  
Therapeutic Enzymes

The enzymes' biocatalysts act by lowering the activation energy without getting consumed in the reaction. The immense number of enzymes acts as a correctly matched orchestra to ensure that enormously complex life mechanisms and processes occur in a right direction. Sufficient quantity and accurate function of enzymes results in proper functional maintenance of body. The enzymes play a major role in the diagnosis, curing, biochemical investigation, and monitoring of many dreaded diseases of the century. The development of recombinant DNA technology had a significant impression on production levels of enzymes. Around 50% of the enzyme market is covered by recombinant enzymes. Because of development in molecular biology tools, several pharmaceutically enzymes have been identified and are being actively used in the pharmaceutical industry either for diagnostic or treatment. Information on this topic is very insufficient, and thus, the present chapter is an attempt to compile information on the sources, properties and applications of important therapeutic enzymes.

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.

scholarly journals SUBKLONING DAN ISOLASI GEN PENYANDI MIKRONEMA 3 (MIC-3) Toxoplasma gondii ISOLAT LOKAL

Molekul ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. 10
Author(s):  
Diana Indrasanti ◽  
Aris Haryanto ◽  
Wayan T. Artama
Keyword(s):  
Toxoplasma Gondii ◽  
Expression Vector ◽  
Recombinant Dna ◽  
Cell Infection ◽  
Recombinant Dna Technology ◽  
Specific Primers ◽  
Gene Encoding ◽  
E Coli ◽  
Local Isolate ◽  
Microneme Protein

Microneme protein (MIC) is one of proteins that belongs to excretory-secretory antigens (ESAs) of Toxoplasma gondii. Microneme 3 protein (MIC-3) is the protein that plays an important role in the invasion proccess during cell infection as a mediator attachment parasite to the host cell. The aim of this research is to clone mic3 (gene encoding for MIC-3) of T. gondii from local isolate using recombinant DNA technology by cloning mic3 in an expression vector. Deoxyribonucleic acid (DNA) from T. gondii tachyzoites was amplified by PuRe Taq RTG-PCR Beads using mic3 specific primers. Amplified DNA was double digested using EcoRV and HindIII restriction endonucleases and then purified using EZ-10 spin coloumn purification kit. The mic3 DNA was ligated into pET-32a(+) expression vector and transformated into Escherichia coli BL21. The results showed that recombinant mic3gene 4.2 kDa has been successfully performed by cloning gene encoding for MIC-3 protein of T. gondii local isolate into pET-32a(+) and transformed to E. coli BL21.

scholarly journals Enzymes of industrial interest

2017 ◽  
Vol 2 (2) ◽  
pp. 74-97 ◽  
Author(s):  
Arnold L. Demain ◽  
Sergio Sánchez
Keyword(s):  
Recombinant Dna ◽  
Animal Feed ◽  
Strain Improvement ◽  
Major Effect ◽  
Industrial Enzymes ◽  
Recombinant Enzymes ◽  
Recombinant Dna Technology ◽  
Microbial Enzymes ◽  
Wide Range ◽  
The World

For many years, industrial enzymes have played an important role in the benefit of our society due to their many useful properties and a wide range of applications. They are key elements in the progress of many industries including foods, beverages, pharmaceuticals, diagnostics, therapy, personal care, animal feed, detergents, pulp and paper, textiles, leather, chemicals and biofuels. During recent decades, microbial enzymes have replaced many plant and animal enzymes. This is because microbial enzymes are widely available and produced economically in short fermentations and inexpensive media. Screening is simple, and strain improvement for increased production has been very successful. The advances in recombinant DNA technology have had a major effect on production levels of enzymes and represent a way to overproduce industrially important microbial, plant and animal enzymes. It has been calculated that 50-60% of the world enzyme market is supplied with recombinant enzymes. Molecular methods, including genomics and metagenomics, are being used for the discovery of new enzymes from microbes. Also, directed evolution has allowed the design of enzyme specificities and better performance.

scholarly journals Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse

2021 ◽  
Author(s):  
Raquel Guimarães Benevides ◽  
Sandra Aparecida de Assis ◽  
Alison Borges Vitor ◽  
Geise Camila Ribeiro ◽  
Cleidineia Souza de Santana ◽  
...  
Keyword(s):  
Recombinant Dna ◽  
Cellulose Degradation ◽  
Fungal Species ◽  
Biofuel Production ◽  
Chromosomal Dna ◽  
Recombinant Enzymes ◽  
Recombinant Dna Technology ◽  
High Quality ◽  
Efficient Manner ◽  
Enzymatic Production

Cellulases are important enzymes in cellulose degradation that occurs in nature, this degradation involves a system of extracellular multienzymes and have wide application. The construction of a high-quality system for the production of these enzymes is important for its application in the process of saccharification of biomass involved in the biofuel production process. Several species of fungi are capable of synthesizing and secreting high amounts of cellulase, most studies with fungal species use linearized plasmid, since these are encompassed to chromosomal DNA, improving its stability and expression efficiency. Advances in the production of recombinant enzymes focus on the search for industrially viable microorganisms capable of producing enzymes under various conditions, expressing them in a highly efficient manner, aiming at the synthesis of several copies of genes and a strong promoter. To resay these restrictions, molecular biology combined with recombinant DNA technology is a viable tool in enzymatic production. In subsequent topics, the production of endoglucanases, exoglucanases and β-glucosidase of fungi cloned in Escherichia coli, Pichia pastoris and other different expression systems will be addressed.

Construction of E. coli producer of human interleukin-2 based on recombinant DNA technology

1995 ◽  
Vol 31 ◽  
pp. 364
Author(s):  
L.G. Glushakova ◽  
V.I. Prima ◽  
E.N. Kovalchuk ◽  
V.A. Kordyum
Keyword(s):  
Recombinant Dna ◽  
Interleukin 2 ◽  
Recombinant Dna Technology ◽  
E Coli ◽  
Dna Technology

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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