Gene synthesis and expression in E. coli for pump, a human matrix metalloproteinase.
The gene for PUMP (putative metalloproteinase), a human matrix metalloproteinase, was synthesized by a PCR-based method. The DNA fragment of 546 bases containing the PUMP gene was generated by overlap extension of six long oligonucleotides (length ranging from 101 to 116 bases) and subsequent amplification by two short terminal oligonucleotide primers (length from 20 to 48 bases) in one pot without using restriction and ligation enzymes. The synthetic gene was cloned into a T7 expression vector in two ways to express PUMP as a non-fusion protein. Both constructs showed high level expression in E. coli.
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Department of Biochemistry, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105.
Johnson
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Baragi
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005813:Genes, Synthetic; D006801:Humans; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D008666:Metalloendopeptidases; D020783:Matrix Metalloproteinase 7