To generate pSK-CP60 plasmid, CP60 cDNA was amplified with primers 5′-ataGAATTCatggcaatccaactggac-3′ (upstream, containing EcoRI site) and 5′-ataGGATCCctgctccagttcgtcg-3′ (downstream, containing BamHI site) from cDNA library. The PCR product was cleaved with BamHI and EcoRI, and the 1330 bp BamHI–EcoRI fragment was cloned into pBluescript II SK + vector cleaved with the same enzymes.
Plasmids for the yeast two-hybrid assay were prepared using the full-sized and truncated versions of CP60 protein, CP190, Mod(mdg4)-67.2, and Su(Hw) fused with pGAD424 and pGBT9 vectors (Clontech, Mountain View, CA, USA). Details of the cloning procedures are described in theSupplementary Materials .
Plasmid for expression CP60 protein in E. coli was generated by cloning the BamHI (filled in with Klenow fragment)–EcoRI fragment from pSK-CP60 into pET32a cleaved with NotI (filled in with Klenow fragment) and EcoRI. Expression constructs for Su(Hw) and CP190 proteins were described previously [58 (link),59 (link),60 (link),66 (link)]. Plasmid for expression Adf1 protein was kindly provided by Dr. Maxim Erokhin [80 (link)].
Plasmids for the yeast two-hybrid assay were prepared using the full-sized and truncated versions of CP60 protein, CP190, Mod(mdg4)-67.2, and Su(Hw) fused with pGAD424 and pGBT9 vectors (Clontech, Mountain View, CA, USA). Details of the cloning procedures are described in the
Plasmid for expression CP60 protein in E. coli was generated by cloning the BamHI (filled in with Klenow fragment)–EcoRI fragment from pSK-CP60 into pET32a cleaved with NotI (filled in with Klenow fragment) and EcoRI. Expression constructs for Su(Hw) and CP190 proteins were described previously [58 (link),59 (link),60 (link),66 (link)]. Plasmid for expression Adf1 protein was kindly provided by Dr. Maxim Erokhin [80 (link)].
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