Cloning and Prokaryotic Expression of GDA2 Gene and Purification of GDA2-GST Fusion Protein
Sultan Ababakri1,2 Bai Yong1 Zhu Yuxian1**
(1. National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China; 2. College of Life Sciences and Technology, Xinjiang University, Urumqi 830046, China)
Abstract：Abstract: GDA2 is one of the senescence-related genes that were cloned from G2 pea (Pisum sativum L.) previously. It shares 36% sequence identity with the previously reported GDA1 gene. This cDNA was amplified by PCR method from the original cloning vector, pBSK-GDA2, and then cloned into the T-easy-vector to produce the new construction, pGEM-T-GDA2. Sequence analysis showed that the cloned cDNA of GDA2 is 1 120 bp long, which has an open reading frame of 642 bp and encodes a protein of 213 amino acids with a calculated molecular weight of 24 kD. By PCR method, Bgl Ⅱand XhoⅠ restriction sites were added to the 5' and the 3' end of the GDA2 respectively. The expression vector pGEX-GDA2 was constructed by inserting the coding region of GDA2 cDNA into pGEX 4T-1. And the IPTG-inducible product, a fusion protein which consists of 27 kD GST protein and 24 kD GDA2 protein at C-terminal, was finally purified by Glutathione Sepharose 4B affinity column.
