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.
Moguilevsky
N
N
Applied Genetics, University of Brussels, Nivelles, Belgium.
Varsalona
F
F
Guillaume
J P
JP
Gilles
P
P
Bollen
A
A
Roobol
K
K
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D004274:DNA, Recombinant / Q000235:genetics; D004798:Enzymes; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005069:Evaluation Studies as Topic; D006801:Humans; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011498:Protein Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D004274:DNA, Recombinant; D004798:Enzymes; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D008715:Methionine