Effectivity of expression of mature forms of mutant human apolipoprotein A-I

In order to probe the structural and functional properties of a central reg ion of apolipoprotein A-I (apoA-I), we engineered mutants of the mature for m of the protein and expressed them using the baculovirus/ insect cell expr ession system. The mutations which targeted the region of apoA-I between am ino acids 140 and 150 included: (i) deletion of the region 140-150 (apoA-I( Delta 140-150)); (ii) substitution of arginine 149 with valine (apoA-I(R149 V)); (iii) substitution of proline 143 with alanine (apoA-I(P143A)); (iv) d eletion of region 63-73 (apoA-I(Delta 63-73)), which has structural propert ies 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 efficiencie s of synthesis were vastly different for the various mutants as follows: ap oA-I(R149V) > apoA-I(140-150 i 63-73) > apoA-I(Delta 63-73) > apoA-I(P143A) > apoA-I > apoA-I(Delta 140-150). About 50% of the synthesized wild type a nd 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 pro duced 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-a poA-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 r esults demonstrate that at least in the model of insect cells, the efficien cy of apoA-I synthesis, processing, and secretion depends on apoA-I seconda ry structure and/or folding. (C) 1999 Academic Press.