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: email@example.com, firstname.lastname@example.org
Expression analysis of hEGF on 15% polyacrylamide gel. A, Expression of hEGF for single copy construct. Lane 1, PAGE Ruler TM unstained protein ladder. Lane 2, Un-induced sample. Lanes 3 to 7 show induced samples having bands of hEGF slightly below than 10kDa are clearly visible. B, Expression of hEGF for double copy construct. Lane 1, PAGE Ruler TM unstained protein ladder. Lane 2: Un-induced sample. Lanes 3 to 7: Induced samples. Bands of hEGF with much bright intensity are clearly visible. C, Expression of hEGF for triple copy construct. Lane 7: PAGE Ruler TM unstained protein ladder. Lane1, Un-induced sample. Lane 2 to 6, Induced samples. Bands of hEGF are visible. Intensity of bands were analyzed by densitometric analysis.
Showing relative expression level of human epidermal growth factor in 3 different populations of Rosetta-gamiTM 2 (DE3) strain having single, double and triple copies of expression cassette. The mean value for expression level in clones containing pET28-EGF-C1 was found to be 0.61 ± 0.04. The mean value for expression level in clones containing pET28-EGF-C2 was found to be 2.36 ± 0.21 which is 3.8 folds higher as compare to their counterparts having single expression cassette. The clones containing expression vector with three expression cassettes i.e. pET28-EGF-C3 showed the mean expression level of 1.94 ± 0.154 which is 3.1 folds higher as compare to their single copy counterparts. The error bars indicate the standard error for each population.
Western blotting for confirmation of expression of human epidermal growth factor in clones having expression cassettes. Lanes 1-3, Bands appeared on nitrocellulose membrane after NBT/BCIP treatment showing successful expression of hEFG in clones containing pET-EGF-C1, pET-EGF-C2 and pET-EGF-C3 respectively. Lane 4, Un-induced sample used as negative control. No band appeared for negative control.
Maps of single, double and triple copy constructs prepared for the expression of human epidermal growth factor by multimerization of hegf gene. The expression cassette of about ~340bp was released by digestion with Bgl II and BamHI from single copy construct Figure 1A and inserted back to its BamHI linearized form for preparation of double copy construct i.e., pET28-EGF-C2 as shown in Figure 1B. The same expression cassette was inserted to the backbone of double copy construct, linearized with BamH1, for preparation of triple copy constructsi.e. pET28-EGF-C3 as shown in Figure 1C. Sequential steps repeated in same pattern can generate constructs with multiple copies of genes. Sequence maps were generated using SnapGene software version for Windows.