Production of authentic human proapolipoprotein A-I in Escherichia coli: strategies for the removal of the amino-terminal methionine.

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 recombinant 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 enterokinase, 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 peptide of the bacterial outer membrane protein, OmpA, aiming at an in situ conversion to authentic proapo A-I during secretion to the bacterial periplasm. The data showed that, out of these five systems, the OmpA signal peptide system and, to a lesser extent, the one involving the fusion to ubiquitin were the most efficient in yielding authentic proapo A-I from engineered Escherichia coli.