Takao T, Kobayashi M, Nishimura O, Shimonishi Y
J Biol Chem. 1987 Mar 15;262(8):3541-7.
Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.
对在大肠杆菌中大量表达的重组人白细胞干扰素A的生物活性组分进行蛋白水解消化，并用快原子轰击质谱法和高效液相色谱法进行分析。用胰蛋白酶和金黄色葡萄球菌蛋白酶V8对重组人白细胞干扰素A的主要组分进行消化，其质谱中观察到的值占根据编码该蛋白的基因的核苷酸序列预测的人白细胞干扰素A氨基酸序列的93%，这表明重组人白细胞干扰素A的主要组分的氨基酸序列与从编码该蛋白的基因的核苷酸序列翻译而来的氨基酸序列相同。对重组人白细胞干扰素A的两个次要组分的蛋白水解消化产物进行质谱分析，并对其高效液相色谱组分进行质量和氨基酸分析，结果表明干扰素N端氨基酸残基的氨基部分被乙酰化，Cys-1和Cys-98残基被氧化成半胱氨酸或与谷胱甘肽相连。这些发现表明，在大肠杆菌中大量制备的重组蛋白中的氨基酸残基在翻译后被修饰。
