Phospholipase A2의 저해 단백질로서 항염증 치료제로 알려진 인체 lipocortin을 효모로부터 생산하기 위해 인체 lipocortin-1 유전자의 cloning 및 발현 연구를 수행하였다. 인체 lipocortin-1 cDNA를 CYC1, GALl-GALlO 및 PHO5 유전자의 promoter를 갖는 발현 vector에 cloning시킨 후 유전자의 발현을 조사한 결과 각 promoter의 강도 및 사용한 plasmid의 copy수에 따라 발현효율이 달라져 GALl-GALlO promoter와 $2{mu}m$ 기원의 multicopy수의 plasmid를 사용했을 경우 인체 lipocortin-1 유전자의 발현효율이 가장 높음이 확인되었다. 효모 내에서 생산된 인체 lipocortin을 배지로 분비시키기 위해 효모의 ${alpha}$ 교배인자의 pre-pro leader 배열과 인체 lipocortin-1 유전자를 융합시킨 결과 효율적인 post-translational processing을 거쳐 인체 lipocortin만이 일부 분해된 상태로서 체외로 분비됨이 관찰되었다.
Human lipocortin is a phospholipase A2 inhibitory protein and known as a potential antiinflammatory agent. Thus, molecular cloning and expression of human lipocortin-1 gene was tried using a number of yeast expression vectors. cDNA of human lipocortin-1 was placed under the control of CYC1, GAL1-GAL10 and PHO5 promoters. The expression levels were varied according to the promoter strength and the plasmid copy number. It was observed that the human lipocortin-1 gene was able to be expressed under the control of all promoters employed but the highest level of gene expression was achieved by use of the divergent GAL1-GAL10 promoter. The $2{mu}m$ based multicopy number plasmid showed an increased expression level by 50% compared with ARS based single copy number plasmid. To excrete human lipocortin produced in Saccharomyces cerevisiae cells into the culture medium, the pre-pro leader sequence of yeast a mating factor gene was fused with human lipocortin-1 gene. The efficient secretion of the native protein was observed with some extent of degradation in the culture medium after the post-translational processing of the fused protein.
