Over-expression of proteins using a modified pBAD24 vector in E. coli expression system.
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/10(6) c.f.u. of E. coli induced cells while the pBAD24M-SAK vector showed nearly 55 ng/10(6) 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.
Banerjee
Sampali
S
Biotechnology R & D, Lupin Limited, Lupin Research Park, 46A/47A, Nande Village, Mulshi Taluka, Pune, 411042, India.
Salunkhe
Shardul S
SS
Apte-Deshpande
Anjali D
AD
Mandi
Naganath S
NS
Mandal
Goutam
G
Padmanabhan
Sriram
S