Expression of polyisoprenylated Ras proteins in the insect/baculovirus system.

Kus proteins and many related proteins possess the C-terminal motif C‘ys-AAX. where A is an aliphatic ;imino acid and X is itny iiniino acid. ‘I’his sequence directs ii prenyltransferiise. fh-nesyltransferase in the c;ise of ltas and geranylger;inyItransferase in most other ltas-related proteins. t o prenylate the cysteine residue 11-01. The nitture of the amino acid residue X appears t o determine uhether the protein is ii substriite for farnesyliition or geranylgeriinylation 15 I . A protease, the properties of which hiive not been clearly defined, removes the three Cterminal amino acids leaving the prenylated cysteine ;it the C-terminus. The free carboxyl group is then carboxylmethylated by ii carboxylmethyltransterase, though it is not knou II uhether this is fully stoichiometric 17. X I. ‘I‘he Itits family of proteins can be divided into t\\ o sub-groups depending upon the niiture o f sequences N-termin;il of the C‘ys-AAX motif I ] . In one class, exemplified by Kirsten-Hiis (Ki-Itas) (-111) and k i p 1 a, a polybasic region uith clusters of I,ys/Arg residues is present. I n these no further modifications occur. In the other c h s , typified by I Iarvey-Itas ( I Ia-Kas) and N-ltas, further cysteine residues are present. These are sites for I’almitoylatioii. These modifications, in particular the isoprenylation, ilre critical to the biological activity of the protein I 1-4, 0, 10 I. I,;trge quantities of high quality soluble recombinant Kas proteins can be produced in prokaryotic expression systems. I lowever, the protein produced is not modified by lipids. In contrast, the insect/baculovirus expression system carries out a wide range of post-translational modifications typical of eukaryotic cells [ 1 I , 121. An additional