Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization.
A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243. The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable lambda PL promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins. The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping. In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase. The data show for the first time that proapo A-I can be produced efficiently in E. coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product.
Moguilevsky
N
N
Service de Génétique Appliquée, Université Libre de Bruxelles, Nivelles, Belgium.
Roobol
C
C
Loriau
R
R
Guillaume
J P
JP
Jacobs
P
P
Cravador
A
A
Herzog
A
A
Brouwers
L
L
Scarso
A
A
Gilles
P
P
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D011498:Protein Precursors; D011994:Recombinant Proteins; D004247:DNA; D007862:Phosphatidylcholine-Sterol O-Acyltransferase