효모 Saccharomyces cerevisiae의 맞춤형 분비융합인자 기술을 이용한 인체 interleukin-32의 분비생산 연구

Abstract

Yeast S. cerevisiae has long been GRAS organism and used as a host for the production of human therapeutic proteins. Although it has many advantages for the production of human proteins, the secretion level varies considerably according to the type of proteins. To minimize the inconsistency between proteins and improve the secretion level of “difficult-to-express” proteins, we, previously, developed a high throughput screening system for the optimal translational fusion partner (TFP) by an invertase-based autoselection system. To screen an optimal TFP for the secretory production of a novel human cytokine IL-32 which is an inducer of TNFα, IL-32α, IL-32β and IL-32γ gene were inserted into vectors containing 5x103 TFP library and invertase gene (SUC2) via in vivo recombination. For IL-32α, over two hundred transformants and IL-32β, 16 transformants and IL-32γ, 11 transformants appeared on a medium containing sucrose as a sole carbon source. Removal of SUC2 and insertion of IL-32termination codon into the vector library isolated from all transformants were performed by PCR, simultaneously. After retransformation of PCR product and vector fragment into yeast, for IL-32α, randomly selected 40 transformants were tested for their secretion of IL-32 in culture supernatant. Five different TFPs were simply obtained for the optimal secretion of IL-32. Among them, TFP3 was selected for the optimal secretion of IL-32α. Fed batch fermentation of selected strain revealed the secretion of IL-32a up to 300 mg/liter into culture supernatant. Three different TFPs were obtained for IL-32β and 2 different TFPs were obtained for IL-32γ. Selected TFPs could also improve the secretion level of IL-32β and IL-32γ, respectively. Each recombinant strain developed in this study will be useful for the production of three novel cytokines, IL-32α, β and γ.