Abstract
A modified pBAD24 vector (pBAD24M) was constructed with the araBAD promoter of the arabinose operon along with T7g10 sequence elements and a modified Shine–Dalgarno sequence. While both green fluorescent protein and granulocyte colony stimulating factor showed negligible expression under the original pBAD24 vector, they were expressed at >35% of total cellular protein with the modified vector. Similar results were obtained for staphylokinase wherein the pBAD24-SAK construct yielded 8 ng/106 c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/106 c.f.u. induced bacterial cells as tested by ELISA. Interestingly, the expression levels using modified pBAD24 vector matched that achieved with T7 promoter based vector system. The modified pBAD24 vector therefore represents a simple and a useful prokaryotic expression system for efficient repression, modulation and elevated protein expression levels.
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Acknowledgments
The authors wish to thank Dr. Kamal Sharma, Managing Director, Lupin Limited, India for his constant support and encouragement. Authors also thank Dr. Seiichi Yasuda, National Institute of Genetics, Japan for providing pBAD24 vector as a gift.
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Banerjee, S., Salunkhe, S.S., Apte-Deshpande, A.D. et al. Over-expression of proteins using a modified pBAD24 vector in E. coli expression system. Biotechnol Lett 31, 1031–1036 (2009). https://doi.org/10.1007/s10529-009-9976-6
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DOI: https://doi.org/10.1007/s10529-009-9976-6