title: Effectivity of expression of mature forms of mutant human apolipoprotein A-I.
abstract: In order to probe the structural and functional properties of a central region of apolipoprotein A-I (apoA-I), we engineered mutants of the mature form of the protein and expressed them using the baculovirus/insect cell expression system. The mutations which targeted the region of apoA-I between amino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I(Delta140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) deletion of region 63-73 (apoA-I(Delta63-73)), which has structural properties similar to 140-150; and (v) a chimeric protein substituting amino acids 140-150 with amino acids 63-73 (apoA-I(140-150 --> 63-73)). The efficiencies of synthesis were vastly different for the various mutants as follows: apoA-I(R149V) > apoA-I(140-150 --> 63-73) > apoA-I(Delta63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta140-150). About 50% of the synthesized wild type and all apoA-I mutants was retained in the cells. During expression of apoA-I(R149V) an unusual spontaneous recombination occurred. In addition to the expected mutant, another form of apoA-I with an apparent M(r) of 36K was produced which consisted of a duplication of the amino-terminal end of apoA-I, from the prepeptide through to amino acid 62, linked to the original pre-apoA-I(R149V) sequence via a 4-amino-acid linker. Despite the fact that this form of apoA-I carries two prepeptides and consequently two cleavage sites, there was little, if any, cleavage at the internal cleavage site. During expression, less than 20% of this mutant was retained in the cells. These results demonstrate that at least in the model of insect cells, the efficiency of apoA-I synthesis, processing, and secretion depends on apoA-I secondary structure and/or folding.
authors.affiliation: Baker Medical Research Institute, Melbourne, Victoria, 8008, Australia. Dmitri.Svridov@Baker.edu.au
authors.forename: D
authors.initials: D
authors.lastname: Sviridov
authors.forename: A
authors.initials: A
authors.lastname: Luong
authors.forename: L
authors.initials: L
authors.lastname: Pyle
authors.forename: N
authors.initials: N
authors.lastname: Fidge
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D017433:Protein Structure, Secondary / Q000235:genetics; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship
chemical_list: D016632:Apolipoprotein A-I