Schellenberger V, Pompejus M, Fritz H J
Institute for Molecular Genetics, Georg August University, Göttingen, Germany.
Int J Pept Protein Res. 1993 Apr;41(4):326-32. doi: 10.1111/j.1399-3011.1993.tb00447.x.
We describe a new approach for the production of peptides using a combination of recombinant DNA technology, chemical synthesis, and proteinase-catalyzed processing. An artificial substance P-precursor is produced as a beta-galactosidase (1-459) fusion protein containing nine copies of the decapeptide sequence Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe. The fusion protein accumulates in E. coli as insoluble inclusion bodies which are easily isolated and purified. The decapeptide blocks are selectively cleaved from the insoluble fusion protein by alpha-chymotrypsin. Alternatively, a dodecapeptide ester is produced when a dipeptide ester is included in the chymotrypsin reaction mixture. This peptide ester is converted converted to substance P by papain-catalyzed acyl transfer and subsequent tryptic cleavage. These results demonstrate that peptides can be readily produced by a combination of recombinant DNA technology and proteinase-catalyzed conversion. The approach allows incorporation of groups other than natural amino acids into oligo- and polypeptides.
我们描述了一种使用重组DNA技术、化学合成和蛋白酶催化加工相结合的方法来生产肽。一种人工P物质前体作为β-半乳糖苷酶（1-459）融合蛋白产生，该融合蛋白包含十个拷贝的十肽序列Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe。融合蛋白以不溶性包涵体的形式在大肠杆菌中积累，易于分离和纯化。通过α-胰凝乳蛋白酶从不溶性融合蛋白中选择性切割十肽片段。或者，当在胰凝乳蛋白酶反应混合物中包含二肽酯时，会产生十二肽酯。该肽酯通过木瓜蛋白酶催化的酰基转移和随后的胰蛋白酶切割转化为P物质。这些结果表明，通过重组DNA技术和蛋白酶催化转化相结合可以很容易地生产肽。该方法允许将天然氨基酸以外的基团掺入寡肽和多肽中。
