Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Recombinant Human Proinsulin: A New Approach in Gene Assembly and Protein Expression

  • Mergulaho, Filipe J.M. (Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, Av. Rovisco Pais, Portugal) ;
  • Monteiro, Gabriel A. (Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, Av. Rovisco Pais, Portugal) ;
  • Kelly, Andrew G. (Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, Av. Rovisco Pais, Portugal) ;
  • Taipa, Maria A. (Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, Av. Rovisco Pais, Portugal) ;
  • Joaquim, M.S. Cabral (Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, Av. Rovisco Pais, Portugal)
  • Published : 2000.10.01
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Abstract

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 mg per liter culture, expressed as inclusion bodies. Protein secretion to the culture medium and periplasmic space was achieved by cloning in the pEZZ18 vector.

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References

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