TABLE 1.
Construct | Vector | Primer pair (5′ region/ 3′ region) | Cloning sites | Mutation |
---|---|---|---|---|
pBac/C2 | pFastBacHTa | PP141/PP142 | NcoI, EcoRI | Wild type |
pBac/C2-C36R | pFastBacHTa | PP141/PP142 | NcoI, EcoRI | Cys36→Arg |
pBac/C2-C38N | pFastBacHTa | PP141/PP143 | NcoI, BamHI | Cys38→Asn |
pBac/C2-C46I | pFastBacHTa | PP141/PP143 | NcoI, BamHI | Cys46→Ile |
pEHT/C2 | pEHISTEV | PP141/PP142 | NcoI, EcoRI | Wild type |
pEHT/C2-C36R | pEHISTEV | PP141/PP142 | NcoI, EcoRI | Cys36→Arg |
pEHT/C2-C38N | pEHISTEV | PP141/PP143 | NcoI, BamHI | Cys38→Asn |
pEHT/C2-C46I | pEHISTEV | PP141/PP143 | NcoI, BamHI | Cys46→Ile |
The TYLCV-C C2 gene and mutant derivatives were PCR amplified using PVX/C2, PVX/C2-C36R, PVX/C2-C38N, and PVX/C2-C46I (37) as DNA templates together with primer PP141 (cctgtatcATGaGATCTTCGTCTCCCTC) and either PP142 (ccgaatTcAAATACTCTTAAGAAATGCGAGGTC) or PP143 (ccggatccTAAATACTCTTAAGAAATGCGAGGTC) (introduced restriction endonuclease sites are underlined and modified nucleotides are lowercase). PCR products were digested with BspHI plus EcoRI or BamHI and were cloned into the NcoI and EcoRI or NcoI and BamHI sites of pEHISTEV (a modified version of pET28a [unpublished data]) and pFastBacHTa (Invitrogen Life Technologies) to produce vectors for C2 protein expression in E. coli and insect cells, respectively.