Nishi S, Koyama Y, Sakamoto T, Soda M, Kairiyama C B
Department of Biochemistry, Hokkaido University School of Medicine.
J Biochem. 1988 Dec;104(6):968-72. doi: 10.1093/oxfordjournals.jbchem.a122592.
Rat alpha-fetoprotein (AFP) cDNA spanning the complete coding region was cloned and expressed in Escherichia coli as well as in yeast, Saccharomyces cerevisiae. The recombinant AFPs (rAFPs) were purified and characterized. The molecular weights determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were 65,000 for E. coli rAFP and 69,000 for yeast rAFP. Amino acid and N-terminal sequence analyses indicated that yeast cells produced mature AFP by processing the signal peptide properly but E. coli rAFP lacked the N-terminal 53 amino acid residues of preAFP. The yeast rAFP was found to be indistinguishable from authentic AFP in the Ouchterlony immunodiffusion test, radioimmunoassay and estradiol-binding assay while E. coli rAFP was less reactive in these tests. These observations indicated that the rAFP expressed in yeast emulated the properties of authentic AFP.
跨越完整编码区的大鼠甲胎蛋白（AFP）cDNA被克隆，并在大肠杆菌以及酿酒酵母中表达。对重组AFP（rAFP）进行了纯化和特性鉴定。通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳测定的分子量，大肠杆菌rAFP为65,000，酵母rAFP为69,000。氨基酸和N端序列分析表明，酵母细胞通过正确加工信号肽产生成熟的AFP，但大肠杆菌rAFP缺少前AFP的N端53个氨基酸残基。在双向免疫扩散试验、放射免疫测定和雌二醇结合试验中，发现酵母rAFP与天然AFP没有区别，而大肠杆菌rAFP在这些试验中的反应性较低。这些观察结果表明，在酵母中表达的rAFP模拟了天然AFP的特性。
