Cloning, Prokaryotic Expression and Purification of CpfS1 Gene from Arabidopsis Thaliana

摘要

CpfS1 Gene cloned from arabidopsis thaliana was expressed in Escherichia coli DH5a. A cDNA fragment about 320 bp was amplified from the total RNA of arabidopsis thaliana seeds by reverse transcription PCR (RT-PCR) with a pair of specific primers based on the sequences of the AtCpfS1 gene. The recombinant prokaryotic expression vector pET30a-AtCpfS1 was constructed by inserting the cDNA fragment encoding the mature peptide into the prokaryotic expression vector pET30a, and then transformed into E. coli DH5a. Sequence analysis showed that the fragment length was 346 bp containing a full coding region of 332 bp encoding 76 amino acid residues with a molecular mass of 21.5 kD. The SDS-PAGE electrophoresis analysis showed that the best expression was induced by 21 degrees C and 3.6x10(-3) mol/L IPTG, under which a relative molecular weight of 82.5 kD recombinant protein was produced. The nickel chelating resin was used to purify the protein in size exclusion chromatography (SEC) and the results indicated that AtCpfS1 protein was present in the form of tetramer.