High-level expression of human fibroblast growth factor-9 N33 in Escherichia coli

Abstract

We constructed plasmids carrying the human fibroblast growth factor-9 N33 (hFGF-9 N33) gene under the control of the T7 promoter in pBR322. These plasmids were introduced into Escherichia coli MM294 lysogenized with bacteriophage λ with the T7 RNA polymerase gene under the control of the lacUV5 promoter. We compared the expression level of hFGF-9 N33 in the transformed E. coli MM294(DE3) pETGAF25 and MM294(DE3) pTG931 which are ampicillin- and tetracycline-resistant strains, respectively. Twice as much hFGF-9 N33 was produced by MM294(DE3) pTG931 as by MM294(DE3) pETGAF25 , although the growth rates of these strains were similar. The expression plasmid pTG941, a derivative of pTG931, was constructed by reduring the size of the long non-coding region between the stop codon of the hFGF-9 N33 gene and the T7 terminator. The amount of hFGF-9 N33 expressed in MM294(DE3) pTG941 was twice that expressed in MM294(DE3) pTG931 , although the growth rates of these strains were similar. The expression level reached a maximum when 10 mg/ l of isopropyl β- d-thiogalactopyranoside, an inducer, was added to the culture at 250 Klett units of growth. Feeding glucose to the culture increased the final growth level and caused a 40% increase in the amount of hFGF-9 N33 produced. Over 0.5 g/ l of hFGF-9 N33 was produced in a large-scale culture by improving the expression plasmids and optimizing the culture conditions.