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Summer Research Fellowship Programme of India's Science Academies

Expression and Purification of GST tagged Histidine Kinase proteins of Rice (Oryza sativa L.) using Affinity Chromatography

Rahul Malakar

Department of Botany, Gauhati University, Guwahati 781014

Prof. Ashwani Pareek

Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067

Abstract

The two components system is one of the most important signaling mechanisms in both prokaryotes and eukaryotes. In plants, it is well developed and consists of three proteins - Histidine Kinases (HKs), Histidine Phosphotransfer proteins (HPTs) and Response Regulator (RR). Histidine Kinases play an important role in signal transduction and generally, they are homo- and heterodimers involved in autokinase, phosphotransfer and phosphatase activity. Here, we are focusing on purification of an important Histidine Kinase protein through the Glutathione S-transferase (GST) Fusion Protein Purification system. This system is a versatile system for the expression, purification, and detection of GST-tagged proteins produced in Escherichia coli (E. coli). A series of site-specific proteases for GST-tag cleavage complements the system. Expression in E. coli yields tagged proteins with the GST moiety at the amino terminus and the protein of interest at the carboxyl terminus. Purification of GST-tagged proteins is based on the affinity of GST to the glutathione ligand coupled to a matrix. The binding of a GST-tagged protein to the ligand is reversible, and the protein can be eluted under mild, non-denaturing conditions by the addition of reduced glutathione to the elution buffer. The technique thus provides a mild purification process that does not affect a protein’s native structure and function. For this work, at first I am learning different techniques related to the project .The techniques that learned in the previous four weeks are – Isolation of plasmid DNA, PCR, Agarose gel electrophoresis, SDS-PAGE, Bacterial transformation, Gene cloning and preparation of different buffers related to the work. In this project, the main objective is the purification of GST-tagged Histidine Kinase protein of rice. Different research papers have been studied related to the protein purification process. In this report, purification of Histidine Kinases (HKs) proteins of rice using GST Protein purification protocol with the help of Affinity Chromatography is discussed.

Keywords: Two Components System (TCS), Signal transduction, Glutathione S-Transferase (GST), Escherichia coli (E. coli), Affinity Chromatography

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