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KMID : 0545120000100050690
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 5 p.690 ~ p.693
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Recombinant Human Proinsulin
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MERGULHA～OM FILIPE J. M.
MONTEIRO, GABRIEL A./KELLY, ANDREW G./TAIPA, MARIA A./CABRAL, JOAQUI, M. S.
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Abstract
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Efficient intron deletion with the correct splicing of the two exons of the human proinsulin gene was accomplished by a novel stepwise method using genomic DNA [5]. The two exons were separately amplified in two steps, using the second step primers that incorporated additional bases complementary to the other exon. The fragments were combined in a third PCR reaction. Cloning and sequencing of the PCR product demonstrated the correct splicing of the two exons. Expression studies, using the pET9a vector, revealed a protein band with the correct size with respect to human proinsulin as confirmed by SDS-PAGE and Western blot. Proinsulin concentration was estimated to be around 200㎎ per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZl8 vector.
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