Purification Of RACK1 And G Protein β Subunit From Arabidopsis And Their Possible Interaction

Protein-protein interactions plays key role in the regulation of many cellular processes, such as gene replication, transcription, protein translation, modification and secretion. It also regulates the cell cycle process, signal transduction and metabolization.Therefore, identification of protein-protein interactions is crucial for understanding cellular functions.Receptor for Activated C Kinase 1 (RACK1) is a versatile scaffold protein which plays critical roles in multiple developmental processes. Heterotrimeric GTP-binding protein is a kind of protein with important physiologically regulatory functions in living cells. RACK1 and G proteinβsubunit (Gβ) protein all belong to WD-40 repeat proteins. A lot of previous results showed that, in mammal cells, RACK1 interacts with Gβ. RACK1 and G proteins have also been identified in higher plants and are proposed to play regulatory roles in physiological, biochemical processes and in the responses to environmental stress etc. However, we still know little whether RACK1 interacts with Gβ(AGB1) in plants like the case in mammals.In this study, we firstly use the BL21 expression system to express Arabidopsis RACK1 and AGB1, and screen the optimal expression conditions, respectively. And then, we use both in vitro (GST pull-down) and in vivo (yeast two hybrid) technique to study the possible interaction between RACK1 and AGB1.The genes encoding RACK1 and AGB1 from Arabidopsis genome were cloned into plasmids pGEX-6P-1 and pET32a, respectively and constructed the expression plasmid pGEX-6P-1/AtRACK1 and pET32a/AGB1. The optimal concentration of IPTG, an inducer of the recombinant plasmids is 0.1mM for both the expression plasmids. The optimal expressing temperature is 24℃. The results also showed that the maximal expression amount in the recombinant plasmids is about 32% and 35% of total expressed protein for RACK1 and AGB1, respectively. Furthermore, about 50%RACK1 protein is expressed in the soluble fraction, whereas the AGB1 is expressed in the inclusionbody. Through a series of purification procedures, including differential centrifugation, affinity chromatography and SDS-PAGE, the purity of RACK1 and AGB1 are 95% and 88%, respectively. There is no interaction between AtRACK1 and AGB1 identified by GST pull down and Yeast-two-Hybrid.