Purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in Escherichia coli

Protein Expression and Purification
R BoismenuR A Murgita

Abstract

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear. A more complete analysis of the physiological activities of this oncofetal protein has, until now, been severely limited by the lack of an appropriate source from which to obtain pure AFP in any sizeable quantity. In the present investigation, we obviate this problem by cloning and efficiently overexpressing mature mouse and human AFP cDNA's in Escherichia coli. For recombinant 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 by the addition of N- and C-terminal encoding oligonucleotides. Mouse AFP cDNA was expressed directly as a full-length molecule in vector pTrp4 or as fusion proteins in plasmids pMALc and pRX1 under the transcriptional control of trp or tac promoters. Accumulation of rMoAFP was significantly increased in protease-deficient E. coli strains over nonprotease-deficient strains, > or = 10% of total cell protein. Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant prote...Continue Reading

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Citations

May 1, 2008·Journal of Drug Targeting·Galina A PosypanovaEvgeny S Severin
May 8, 2009·Journal of Chromatography. a·Yu Chen, Susanna Su Jan Leong
Sep 30, 2009·British Journal of Pharmacology·A I PotapovichL G Korkina
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Jun 15, 2013·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·A A Terentiev, N T Moldogazieva

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