Nucleic Acids Research, 1989, Vol. 17, No. 5 1933-1951
© 1989
MOLECULAR BIOLOGY |
Use of synthetic ribosome binding site for overproduction of the 5B protein of Insertion sequence IS5
Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine Baltimore, MD 21205, USA
*To whom correspondence should be addressed
Received November 21, 1988. Revised February 6, 1989. Accepted February 6, 1989.
Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Although there was no detectable expression from the 5B open reading frame when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. B-galacto-sidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80–85% of the total protein made in vitro and 20–25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vitro Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.
+Present address: E.I.du Pont de Nemours and Co., Inc., Central Research and Development Department, Experimental Station E328/l50, Wilmington, DE 19880-0328, USA