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Fig. 6 | BMC Plant Biology

Fig. 6

From: Genome-wide identification of cyclophilin genes in Gossypium hirsutum and functional characterization of a CYP with antifungal activity against Verticillium dahliae

Fig. 6

PPIase activity assays of recombinant GhCYP-3 in vitro. a, GhCYP-3 with SacI and BglII sites was amplified by PCR and cloned into the prokaryotic expression vector pET-32a. b, Expression and purification of recombinant GhCYP-3 in Escherichia coli. Recombinant GhCYP-3 expression was induced with 1 mM IPTG for 3 h. The resulting proteins were separated by 10% SDS-PAGE and analyzed by western blot using His antibody. 34.6 kDa Recombinant TrxA-6Ă—His-S-tag-GhCYP-3 (THS-CYP) protein (arrows) was purified on nickel-NTA agarose columns. M, marker; Lane 1-2, empty vector pET-32a without IPTG; Lane 3-4, pET-32a- GhCYP-3 without IPTG; Lane 5, empty vector pET-32a with IPTG; Lane 6, pET-32a- GhCYP-3 with IPTG. c, A protease-coupled assay was used to measure PPIase activity of recombinant GhCYP-3. The prolyl cis-trans isomerization of the tetrapeptide substrate (Suc-Ala-Phe-Pro-Phe-2,4-difluoroanilide) was reflected by an increase in absorbance at 390 nm. The curves represent isomerization of the Suc-AFPF-pNA substrate over the course of 350 s in the absence of GhCYP-3 (Blank) and in the presence of 200 nM recombinant GhCYP-3 protein. Values represent the mean of three biological replicates

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