Stability and detection of recombinant pre‐pro‐concanavalin A after cytoplasmic expression in Escherichia coli

[摘要]

The cDNA for pre-pro-concanavalin A (pre-pro-Con A) from Canavalia ensiformis was used to construct two cytoplasmic expression vectors: pKconA (no transcription terminator) and pTKconA (containing the cry transcription terminator). The latter produced 2- to 3-fold greater amounts of pre-pro-Con A. This product containing the plant signal can be detected by Western blotting only after electrophoretic transfer in the presence of SDS, indicating reduced solubility. The signal is not removed and pre-pro-Con A is clearly stable after expression in E. coli JM109. The protein is cleaved and ligated as in the plant, in contrast to a recent report.

[学科分类] 生物化学/生物物理

[关键词] Concanavalin A;Legume lectin;Post-translational processing;Recombinant protein stability;Transcription terminator;Western blotting;Canavalia ensiformis;Escherichia coli;Con A;concanavalin A;IPTG;isopropyl-1-thio-β-d- galactopyranoside;RBS;ribosome binding site