Cloning, Expression, Purification And Preliminary Function Studies Of CLAVATA1 Ectodomain And CpTI

Leucine-rich repeat receptor kinases (LRR-RLKs) are probably the most important cell surface receptors in plants. LRR-RLKs play important roles in regulation of plant development, defense responses and crosstalk among hormone signaling pathway. LRR-RLK protein CLV1 is one of the most important members in the CLAVATA signaling pathway. The soluble protein of LRR-RLK family protein CLV1-ECD was obtained by fusion expression with NusA protein from E.coli. Under two kinds of antibiotics'selective pressure, two incompatible plasmids (pET44a-CLV1-ECD and pET48b-CLV3) can exist stably and express heterogonous protein in E.coli. The co-expression can enhance the solubility of CLV1-ECD. This method can avoid the complex process of inclusion body's denaturation and renaturation, and obtain large-scale purified proteins which will be important to investigate CLAVATA signal pathway in plants.CpTI gene was a widely used insect resistance gene in the plant genetic engineering for its high insecticidal activity. The DNA fragment of CpTI gene was amplified through PCR reaction from total nuclear genomic DNA of cowpea. The target fragment was subcloned into PGEX-6p-1 by reaction of BamHI and XhoI to generate fusion with GST which increased the CpTI stability. The fusion protein GST-CpTI successfully purified by affinity chromatography using Glutathione sepharose 4B. The trypsin inhibitor activity was assayed with BApNA as substrate. This work laid foundation for GST-CpTI as a new biological pesticide.