Development Of A Novel T/A Cloning And Expression Vector For Efficient Expression Of Protein-Based Biopharmaceuticals In Escherichia Coli
Loading...
Date
2017-05
Authors
Che Nordin, Muhamad Alif
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Escherichia coli is one of the most popular bacterial hosts for handling, manipulating, and expressing target genes of interest. Certain genes are difficult to express, often due to scarcity of specific tRNA. This has led to little and/or poor quality protein production in E. coli. To alleviate this problem, E. coli hosts are transformed with rare tRNA-expressing helper plasmid vectors. Maintenance of two different plasmid vectors-expressing the gene of interest and rare tRNA genes exert metabolic burden on the host that may result in diminished growth and reduced protein production. To address this issue, we have previously engineered an expression vector that simultaneously expresses rare tRNA genes and the gene of interest. Restriction enzymes are frequently used to clone desired genes into plasmid vectors. However, the presence of restriction enzymes–recognition sites within the desired gene would hamper the cloning process. To this end, we have engineered a rare tRNA vector with a T/A cloning module to facilitate restriction enzyme-independent cloning of genes. This work has resulted in the development of a novel expression vector that featured: a) T/A cloning module for rapid and restriction enzyme–independent cloning; b) three rare tRNA genes for efficient expression of therapeutic proteins; c) a tightly regulated and inducible promoter for controlled expression of the desired gene; and d) a C-terminal hexahistidine tag for rapid purification of recombinant protein.
Description
Keywords
T/A cloning and expression vector for efficient expression , Protein-Based Biopharmaceuticals in Escherichia coli