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Gene Multimerization in Expression Vector: A Potential Strategy for Enhanced Protein Expression in E. coli

Gene Multimerization in Expression Vector: A Potential Strategy for Enhanced Protein Expression in E. coli

Aadil Sultan, Mohsin Ahmad Khan*, Nadeem Ahmed, Muhammad Hassan, Rashid Bhatti, Hafsa Naeem, Muhammad Islam Khan, Saad Tahir,  Samia Afzal* and Ahmad Ali Shahid

Center of Excellence in Molecular Biology, 87-West Canal Bank Road, Thokar Niaz Baig, University of the Punjab, Lahore, Pakistan

*      Corresponding author:,


Protein production in any expression system can be enhanced either by optimizing the culturing parameters or targeting the genetic factors that enhance protein production. One of those genetic factors is gene dosage, which can be increased by increasing the vector copy number. However, increased number of expression vector poses some metabolic burdens on bacterial cells. The current study provides gene multimerization strategy as an alternate way to amplify the gene dosage and explores its effect on the expression of human epidermal growth factor (hEGF) by constructing three types of expression vectors i.e., pET28-EGF-C1, pET28-EGF-C2 and pET28-EGF-C3, each containing single, double and triple expression cassettes, respectively. These vectors were transformed in E. coli strain Rosetta-gami 2 (DE3) to develop three different bacterial populations. Expression of human epidermal growth factor was analyzed by SDS-PAGE and subsequent densitometric analysis was done using ImageJ software. The difference in protein expression of all three populations was analyzed by using one-way ANOVA. Clones with double and triple copies of genes showed an increased expression up to 3.8 and 3.1 folds as compare to the clones having single copy of gene. Gene multimerization strategy can be used to enhance production of recombinant proteins in bacterial expression system.

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Pakistan Journal of Zoology


Vol. 55, Iss. 3, Pages 1003-1500


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