Engineering by PCR-based exon amplification of the genomic porcine interferon-gamma DNA for expression in Escherichia coli.
Interferon-gamma (IFN-gamma) is coded for by a single gene containing three introns, localized within the coding region. We have previously cloned the IFN-gamma gene from a pig genomic DNA lambda library and have determined its nucleotide sequence. In order to construct the porcine IFN-gamma DNA without intervening sequence, the four exons were separately amplified by the polymerase chain reaction (PCR) using primers matching the exon-termini. From the amplified exon-fragments the complete intron-free DNA was obtained by a strategy consisting of alternate rounds of PCR and ligation. The sequence so-obtained was used for expression in E. coli. The recombinant protein appeared as inclusion bodies which were solubilized and refolded in order to obtain biologically active IFN-gamma.
Vandenbroeck
K
K
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium.
Dijkmans
R
R
van Aerschot
A
A
Billiau
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005091:Exons; D015698:Genomic Library; D007371:Interferon-gamma / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D007371:Interferon-gamma; D004247:DNA