PURIFICATION AND CHARACTERIZATION OF HUMAN AND MOUSE RECOMBINANT ALPHA-FETOPROTEINS EXPRESSED IN ESCHERICHIA-COLI

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear. A more complete analy sis of the physiological activities of this oncofetal protein has, unt il now, been severely limited by the lack of an appropriate source fro m which to obtain pure AFP in any sizeable quantity. In the present in vestigation, we obviate this problem by cloning and efficiently overex pressing mature mouse and human AFP cDNA's in Escherichia coli. For re combinant mouse AFP (rMoAFP), large segments of the coding region were excised from the preexisting plasmids pAFP1 and pAFP2, which together encompass 90% of the AFP sequence. The mouse cDNA was made complete b y the addition of N- and C-terminal encoding oligonucleotides. Mouse A FP cDNA was expressed directly as a fall-length molecule in vector pTr p4 or as fusion proteins in plasmids pMALc and pRX1 under the transcri ptional control of trp or tac promoters. Accumulation of rMoAFP was si gnificantly increased in protease-deficient E. coli strains over nonpr otease-deficient strains, greater than or equal to 10% of total cell p rotein. Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant protein stability, overall levels of synthesis, or facilitated purific ation. Recombinant AFP polypeptides expressed by pTrp4 were as expecte d, deposited in bacterial inclusion bodies. Subsequent to resolubiliza tion/refolding, rMoAFP was first enriched by passage over Q-Sepharose resin followed by final purification using immobilized copper-chelate affinity chromatrography. Protein sequencing of the N-terminus reveale d that purified rMoAFP had a deletion of the first nine amino acids co ded for by the full-length mouse AFP cDNA. Similar N-terminal deletion s are observed with AFP isolates originating from natural sources. A c omplete human AFP cDNA was generated from a fetal liver cDNA library a nd was cloned into vector pTrp4. Recombinant human AFP (rHuAFP) was ex pressed under the identical conditions employed for rMoAFP but purific ation had to be modified to include preparative Mono Q anion exchange chromatography. N-terminal sequencing amino acid compositional analysi s, and electrospray mass spectrometry revealed that purified rHuAFP wa s intact and unaltered and that the initiator methionine was completel y removed. The biological activity of recombinant AFP, as judged by it s inhibitory effects on in vitro lymphocyte proliferation, was equival ent to that of the native protein. The availability of large quantitie s of mouse and human recombinant AFP molecules should now permit detai led structure-function analyses of this important oncofetal protein to proceed in a manner unimpeded by previous limitations in both quantit y and quality of the native proteins. (C) 1997 Academic Press.