Efficient expression and purification of soluble Harpin_Ea protein by translation initiation region codon optimization

Harpin_Ea protein can stimulate plants to produce defense responses to resist the attack of pathogens, improve plant immune resistance, and promote plant growth. This has extremely high application value in agriculture. To efficiently express soluble Harpin_Ea protein, in this study, we expressed Harpin_Ea protein with a 6× His-tag in Escherichia coli BL21 (DE3). Because of the low level of expression of Harpin_Ea protein in E. coli, three rounds of synonymous codon optimization were performed on the +53 bp of the translation initiation region (TIR) of Harpin_Ea. Soluble Harpin_Ea protein after optimization accounted for 50.3% of the total soluble cellular protein expressed. After purification using a Ni Bestarose Fast Flow column, the purity of Harpin_Ea protein exceeded 95%, and the yield reached 227.5 mg/L of culture medium. The purified Harpin_Ea protein was sensitive to proteases and exhibited thermal stability. It triggered visible hypersensitive responses after being injected into tobacco leaves for 48 h. Plants treated with Harpin_Ea showed obvious growth-promoting and resistance-improving performance. Thus, the use of TIR synonymous codon optimization successfully achieved the economical, efficient, and soluble production of Harpin_Ea protein.