Thim L, Norris K, Norris F, Nielsen P F, Bjørn S E, Christensen M, Petersen J
Department of Protein Chemistry, Pharmaceuticals Research, Novo Nordisk, Novo Alle, Bagsvaerd, Denmark.
FEBS Lett. 1993 Mar 8;318(3):345-52. doi: 10.1016/0014-5793(93)80543-4.
Recombinant human spasmolytic polypeptide (r-hSP) has been produced in relatively large amounts in Saccharomyces cerevisiae. The two intronless trefoil domains of the hSP-DNA were cloned separately by PCR from human genomic DNA, and the remaining parts of the gene synthesized. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the hSP sequence. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Kex 2 processing enzyme system. The secreted r-hSP was found in a glycosylated and an non-glycosylated form. The two forms of r-hSP were purified from the yeast fermentation broth by a combination of ion-exchange chromatography and preparative HPLC. The overall yield from 8 litres of fermentation broth was 160 mg r-hSP and 219 mg glycosylated r-hSP corresponding to 50% and 34%, respectively. The structure of the r-hSP and the glycosylated r-hSP was determined by amino acid analysis and carbohydrate composition analysis as well as by peptide mapping, amino acid sequencing and mass spectrometric analysis.
重组人解痉多肽（r-hSP）已在酿酒酵母中大量生产。通过PCR从人基因组DNA中分别克隆了hSP-DNA的两个无内含子三叶因子结构域，并合成了基因的其余部分。构建重组质粒以编码由杂交前导序列和hSP序列组成的融合蛋白。前导序列用于将融合蛋白引导到细胞的分泌途径中，并使其暴露于Kex 2加工酶系统。发现分泌的r-hSP有糖基化和非糖基化两种形式。通过离子交换色谱和制备型HPLC相结合的方法从酵母发酵液中纯化出两种形式的r-hSP。8升发酵液的总产率分别为160毫克r-hSP和219毫克糖基化r-hSP，分别对应50%和34%。通过氨基酸分析、碳水化合物组成分析以及肽图分析、氨基酸测序和质谱分析确定了r-hSP和糖基化r-hSP的结构。
