Production of authentic human proapolipoprotein A-I in Escherichia coli Strategies for the removal of the amino-terminal methionine》.pdf

Journal of Biotechnology, 27 (1993) 159-172 159 © 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00 BIOTEC 00806 Production of authentic human proapolipoprotein A-I in Escherichia coli: Strategies for the removal of the amino-terminal methionine Nicole Moguilevsky a, Francesca Varsalona a, Jean-Paul Guillaume a, Pascal Gilles b Alex Bollen a and Kees Roobol c aApplied Genetics, University of Brussels, B-14OO Nivelles, Belgium h UCB-Bioproducts and c UCB-Pharma, Chemin du Foriest, B-1420 Braine-lAlleud, Belgium (Received 7 February 1992; revision accepted 25 March 1992) Summary Several methods were compared with respect to the production of authentic, N-terminal methionine-free proapolipoprotein A-I in engineered Escherichia coli bacteria. A first approach consisted of treating the purified methionylated recom- binant protein with an amino-peptidase, purified from Aeromonas proteolytica. A second series of strategies was based on the construction of proapo A-I encoding cassettes carrying built-in recognition sites suitable for specific in vitro cleavage of the products with kallikrein and cnterokinase, respectively. Along the same line, a fusion between ubiquitin and proapo A-I was produced in E. coli with the prospect to achieve post-purification cleavage with yeast ubiquitin hydrolase. Finally, proapo A-I was fused to the signal peptidc of the bacterial outer mem- brane protein, OmpA, aiming at an in situ conversion to authentic proapo A-I during secr