Fig. 4.

Inhibition of pectin methylesterase (PME) activity by the pepper pectin methylesterase inhibitor (CaPMEI). a Recombinant CaPMEI1 expression in E. coli BL21 (DE3) pLysS. Cells were grown in LB media, and recombinant protein expression was induced with 1 mM IPTG. M, molecular marker (kDa); Lane 1, Uninduced E. coli BL21 cell extracts; Lane 2, Soluble fraction of E. coli BL21 cell extracts encoding thioredoxin protein after IPTG induction; Lane 3, Purified thioredoxin; Lane 4, Crude protein extracts of E. coli cells producing the thioredoxin–CaPMEI1 fusion protein after IPTG induction; Lane 5, Purified thioredoxin–CaPMEI1 fusion protein; Lane 6, Cleaved CaPMEI1 and thioredoxin proteins following enterokinase digestion. Protein staining was performed using Coomassie brilliant blue. b Inhibition of PME activity following treatment with CaPMEI1. For the inhibition assay, crude CaPMEI1 (0.5 μg) was mixed with 7.8 mU of orange peel PME in a total volume of 11 μL, and then preincubated at 25°C for 15 min, followed by addition to the reaction solution