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Abstract(s)
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 X P^ 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.
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Mary Ann Liebert, Inc., Publishers