The pET-Rep recombinant plasmid construction and geminivirus Rep [C1] gene expression in Escherichia coli strain BL21

Abstract

Abstract Geminivirus Rep [C1] gene produces a replication protein [Rep] that interacts with Retinoblastoma-related protein [RBR] when Geminivirus infect the plants. That interactions interfere with RBR and transcription factor function. So the mechanism of chili plant defense against symptoms of Pepper Yellow Leaf Curl Disease is blocked. The NPR1 gene is a transcriptional co-activator involved in the regulation defense mechanism of Systemic Acquired Resistance [SAR] in chili plants. In silico, Rep protein blocks the SAR mechanism that is regulated by the NPR1 gene. This study aimed to obtain a construction of pET-28a+ plasmid expression recombinant Rep [C1] gene and Rep protein that can be expressed in E.coli BL21. This research is useful to study the interaction of Geminivirus with transcription co-activator in chili plants. The construction was done by ligating plasmid and Rep genes which have been cut using restriction enzymes BamHI and SacI. The construction of a recombinant pET-Rep plasmid is confirmed by amplification and verification of the nucleotide sequence through sequencing techniques. Nucleotide sequence verification results proved that the Rep [C1] gene is successfully constructed into a pET-28a+ plasmid with proper position and orientation. The pET-Rep recombinant proceeds to the expression test using the E.coli BL21 host. Expression was performed using the induction method with IPTG. Proteins Rep is purified using the MagnehisTM Protein Purification System. Rep protein is visualized with SDS-PAGE. Visualization showed that Rep protein was successfully expressed in E.coli BL21 where protein size is estimated of 41,03 kDa in size.