Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli |
| |
Authors: | Mohamed A Marahiel Michael Krause and Hans-Jacob Skarpeid |
| |
Institution: | (1) Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstraße 29, D-1000 Berlin 10;(2) Present address: Biokjemisk Institutt, postboks 1041 Blindern, N-0316 Oslo 3, Norwegen |
| |
Abstract: | Summary The entire structural gene for tyrocidine synthetase 1 from Bacillus brevis ATCC 8185 has been cloned and expressed in Escherichia coli. Transformed E. coli cells were screened for their ability to produce tyrocidine synthetase 1 by in situ immunoassay using antibodies against gramicidin S synthetase 2 which cross-react with tyrocidine synthetase 1. The cloned gene is within a 5.2 kb fragment of B. brevis genomic DNA and requires no external promoter for its expression in E. coli. It was also observed that cloning of the 5.2 kb insert in the opposite orientation still resulted in a high level of tyrocidine synthetase 1 expression in transformed E. coli cells. In addition, protein blotting and partial purification of the gene product by gel filtration revealed a major protein of molecular weight about 100,000 with specific d-phenylalanine dependent ATP-32PPi and 2 deoxy ATP-32PPi exchange activities. These unique activities of tyrocidine synthetase 1 were not detected in protein extracts of E. coli strains carrying the vector. |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|