FEMS MicrobiologyLetters 83 (1991) 29-34 © 199l Federation of European Microbiological Societies 0378-1097/91/$03.50 Published by Elsevier ADONIS 037810979100406S 29 FEMSLE 04601 High level production of Echerichia coli outer membrane proteins OmpA and OmpF intracellularly in Bacillus subtilis Ritvaleena Puohiniemi, Sarah Butcher, Eveliina Tarkka and Matti Sarvas National Public Health Institute, Helsinki, Finland Received 6 June 1991 Accepted 11 June 1991 Key words: OmpA production; Escherichia coli; OmpF production; Bacillus subtilis; Outer membrane proteins; Inclusion bodies; Detergent solubilization 1. SUMMARY A high yield of Escherichia coli outer mem- brane proteins OmpA (about 200 mg/1) and OmpF (about 100 rag/l) was obtained in Bacillus subtilis when produced intracellularly. The yield was more than 100-fold higher than the yield of these proteins by a similar vector containing the complete signal sequence of a-amylase of B. amyloliquefaciens. Both proteins isolated after breakage of the B. subtilis cells by low-speed centrifugation were about 70% pure and could be solubilized by Sarkosyl, SDS and guanidine hy- drochloride. 2. INTRODUCTION Outer membrane proteins of Gram-negative bacteria have been a subject of considerable in- terest as potential vaccine candidates and as anti- Correspondence to." R. Puohiniemi, National Public Health Institute, Mannerheimintie 166, SF-00300 Helsinki, Finland. gens for immuno-diagnosis. For these purposes large quantities of purified proteins would be needed, a goal made difficult by their nature as integral membrane proteins and their tight asso- ciation with other outer membrane components, especially endotoxin (lipopolysaccharide, LPS) (see review [1]). Producing them in a heterolo- gous organism which lacks both the outer mem- brane and LPS therefore would offer many ad- vantages. We have recently described the use of the Gram-positive Bacillus subtilis as a host for the synthesis of the outer membrane protein OmpA of Escherichia coli [2,3]. However, the production level obtained was relatively low, about 1 mg/l of culture. In those studies we used an expression and secretion vector which contained the efficient a-amylase promoter of B. amyloliquefaciens fol- lowed by the secretory signal sequence of the same gene. The OmpA expressed from this vec- tor was cell-associated, not secreted, and the sig- nal sequence was not cleaved [2,3]. In this paper we examine the possibility of obtaining a higher yield of OmpA in B. subtilis by deleting the signal sequence and thus directly Downloaded from https://academic.oup.com/femsle/article-abstract/83/1/29/515448 by guest on 27 May 2020