Benito A, Villaverde A
Institut de Biologia Fondamental, Universitat Autònoma de Barcelona, Spain.
FEMS Microbiol Lett. 1994 Oct 15;123(1-2):107-12. doi: 10.1111/j.1574-6968.1994.tb07208.x.
Seven internal, putatively exposed regions of Escherichia coli beta-galactosidase have been explored regarding their tolerance to insertions of large foreign peptides. Small sequence modifications, including amino acid substitutions and small deletions, were introduced into the lacZ gene to generate unique BamHI restriction sites. By using these mutant genes, a 27 amino acid stretch reproducing the hypervariable loop of foot-and-mouth disease virus VP1 protein (site A) was further inserted in predefined regions of the enzyme. Among the 13 resulting engineered proteins only three, carrying sequence modifications within a short region, are active, with only moderate reduction of their specific activities. The identified permissive region, which involves amino acids 275 to 279, seems to be a flexible area that could be appropriate to incorporate and study biological properties of heterologous peptides in correctly folded beta-galactosidase chimeric proteins.
已对大肠杆菌β-半乳糖苷酶的七个内部假定暴露区域对插入大的外源肽的耐受性进行了研究。对lacZ基因进行了小的序列修饰，包括氨基酸取代和小的缺失，以产生独特的BamHI限制性位点。通过使用这些突变基因，将一段27个氨基酸的片段（重现口蹄疫病毒VP1蛋白的高变环，位点A）进一步插入到该酶的预定义区域。在产生的13种工程蛋白中，只有三种在短区域内带有序列修饰的蛋白具有活性，其比活性仅适度降低。所确定的允许区域涉及氨基酸275至279，似乎是一个灵活的区域，适合在正确折叠的β-半乳糖苷酶嵌合蛋白中整合和研究异源肽的生物学特性。
