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

Md Fakruddin,Khanjada Shahnewaj Bin Mannan,Md Nur Hossain,Reaz Mohammad Mazumdar,Abhijit Chowdhury

doi:10.5402/2013/590587

Abstract

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.

Highlights

In the past few years recombinant DNA technology has enabled scientists to produce a large number of diverse proteins, in microorganisms, that were previously unavailable, relatively expensive, or difficult to obtain in quantity [1]
Bacterial expression systems are commonly used for production of heterologous gene products of both eukaryotic and prokaryotic origin [4]. e expression of heterologous proteins in E. coli, which is the bacterial system, is most widely and routinely used
Limitations Using E. coli as Heterogeneous Protein Expression Host ey are (1) inability of E. coli as a prokaryotic to carry out posttranslational modi cation which is typical for eukaryotic; (2) limited ability to carry out extensive disul de bond formation; (3) some proteins are made in insoluble form, a consequence of protein misfolding, aggregation, and intracellular accumulation as inclusion bodies; (4) sometimes sufficient expression may not be observed due to protein degradation or insufficient translation; (5) codon sequence for a speci c amino acid in Eukaryotic is different from Prokaryotic as E. coli. is phenomenon is known as “codon bias” which vastly hampers protein synthesis and gene expression in E. coli [6]

Summary

Introduction

In the past few years recombinant DNA technology has enabled scientists to produce a large number of diverse proteins, in microorganisms, that were previously unavailable, relatively expensive, or difficult to obtain in quantity [1]. 2. Enzyme Production in E. coli e expression of recombinant proteins in cells in which they do not naturally occur is termed heterologous protein production. (i) Starch processing industry (ii) Fermentation industry (i) Brewing industry (ii) Fruit juice production (iii) Animal feed industry (i) Fruit juice processing industry (i) Dairy industry (ii) Vegetable oil industry (iii) Leather industry (i) Production of high-fructose syrups (i) Hydrolysis of milk lactose (ii) Digestive aid (i) Production of cyclodextrins for pharmaceuticals and other industries (i) Production of synthetic penicillin (i) Baking, brewing, confectionary, and fruit industries (4) expression can o en be achieved quite rapidly beginning with an eukaryotic cDNA clone, express the protein in E. coli, and purify in milligram quantities in less than 2 weeks; (5) suitable fermentation technology well established; (6) can generate potentially unlimited supplies of recombinant protein; (7) economically attractive [6] Industrial applications (i) Inclusion in detergent preparations (ii) Cheese making (iii) Brewing/backing industries (iv) Meat/leather industries (v) Animal/human digestive aids. (i) Starch processing industry (ii) Fermentation industry (i) Brewing industry (ii) Fruit juice production (iii) Animal feed industry (i) Fruit juice processing industry (i) Dairy industry (ii) Vegetable oil industry (iii) Leather industry (i) Production of high-fructose syrups (i) Hydrolysis of milk lactose (ii) Digestive aid (i) Production of cyclodextrins for pharmaceuticals and other industries (i) Production of synthetic penicillin (i) Baking, brewing, confectionary, and fruit industries (4) expression can o en be achieved quite rapidly beginning with an eukaryotic cDNA clone, express the protein in E. coli, and purify in milligram quantities in less than 2 weeks; (5) suitable fermentation technology well established; (6) can generate potentially unlimited supplies of recombinant protein; (7) economically attractive [6]

Limitations

Homo sapiens

Findings

Conclusion