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. Author manuscript; available in PMC: 2014 Jun 15.
Published in final edited form as: Insect Mol Biol. 2013 Mar 24;22(3):306–319. doi: 10.1111/imb.12024

Figure 1.

Figure 1

Expression and affinity purification of recombinant (r) Amblyomma americanum tick saliva serine protease inhibitor (AamS6) in Pichia pastoris. The mature AamS6 protein cDNA was cloned into the pPICZα plasmid and elctroporated into X33 P. pastoris strain as described. Positive transformants were selected for methanol utilization on yeast agar plates. Selected colonies were grown in culture to A600 of 1 before inducing rAamS6 expression by daily feeding of cultures with methanol to 5% final concentration. Panel A = daily (lanes 1–5) rAamS6 expression detection by Western blotting using the antibody to c-terminus histidine tag. Panel B = Affinity purified rAamS6 electrophored on a 4–16% gradient native acrylamide gel and silver stained. CW = column wash, EP = column eluted protein. Panel C = Validation of N-glycosylation posttranslational modification of rAamS6. + DE = rAamS6 treated with the deglycosylating PngaseF enzyme, −DE = non-treated rAamS6.