Guisez Y, Tison B, Vandekerckhove J, Demolder J, Bauw G, Haegeman G, Fiers W, Contreras R
Laboratory of Molecular Biology, State University of Gent, Belgium.
Eur J Biochem. 1991 May 23;198(1):217-22. doi: 10.1111/j.1432-1033.1991.tb16004.x.
The coding region of the human interleukin-6 (hIL6) gene was fused to the prepro secretion signal of the alpha-mating factor gene in several yeast host strains. It was found that the KEX-2 protease was unable to cleave the prepro-Lys-Arg-Pro-IL6 sequence, but that unspecific cleavage of the precursor protein had occurred. The prepro-Lys-Arg-Ala-Pro-IL6 sequence, however, was correctly recognized and cleaved by the KEX-2 protease, and IL6 was efficiently secreted into the culture medium. The N-terminal Ala-Pro peptide was removed during processing by wild-type yeast strains, but was retained in a ste13 mutant. IL6 as well as the aberrant proteins were not glycosylated. The transformed cells could secrete up to 30 micrograms/ml IL6. The protein was purified from the medium to homogeneity by ion-exchange chromatography and gel filtration, and had a specific activity of about 2 x 10(8) IU/mg in a proliferation assay.
人白细胞介素-6（hIL6）基因的编码区在几种酵母宿主菌株中与α-交配因子基因的前原分泌信号融合。发现KEX-2蛋白酶无法切割前原-Lys-Arg-Pro-IL6序列，但前体蛋白发生了非特异性切割。然而，前原-Lys-Arg-Ala-Pro-IL6序列被KEX-2蛋白酶正确识别并切割，IL6被有效分泌到培养基中。N端Ala-Pro肽在野生型酵母菌株处理过程中被去除，但保留在ste13突变体中。IL6以及异常蛋白未被糖基化。转化细胞可分泌高达30微克/毫升的IL6。通过离子交换色谱和凝胶过滤从培养基中纯化该蛋白至均一性，在增殖试验中其比活性约为2×10⁸ IU/mg。
