Production of GST–SOD fusion protein by recombinant E coli XL1 Blue

A recombinant plasmid was constructed by inserting a DNA fragment with the coding region of Cu/Zn–superoxide dismutase (Cu/Zn–SOD) cDNA from sweet potato, Ipomoea batatas (l) Lam cv Tainong 57, into the 3′ end of the open reading frame of the glutathione S‐transferase (GST) gene in an expression vector, pGEX‐2T. The constructed plasmid was transformed into E coli XL1 Blue. Fusion proteins of Cu/Zn–SOD and GST (GST–SOD) were produced from the recombinant E coli. About 6 mg of GST–SOD fusion proteins could be obtained from 1 dm³ of cultural broth after induction with 0.075 mmol dm⁻³ Isopropyl‐β‐D ‐thiogalactoside (IPTG). Lactose was not an efficient inducer. High cell density culture was performed by fed‐batch fermentation using a glucose analyzer to control glucose concentration at 1 g dm⁻³. The cell density of the fed‐batch culture reached an OD₆₀₀ of 30, the total amount of GST–SOD fusion protein was 100 mg dm⁻³ which is about 14 times more than that of the batch culture. Most of the fusion proteins were shown to be in an active monomeric form, and the molecular weight was estimated to be 45 kDa by SDS–PAGE and 47 kDa by gel filtration. The specific activity of the purified fusion proteins was about 1200 mg⁻¹ and equal to 3200 unit per mg of SOD domain only. © 2000 Society of Chemical Industry