title: Cloning, Prokaryotic Expression and Purification of CpfS1 Gene from Arabidopsis Thaliana.
abstract: CpfS1 Gene cloned from arabidopsis thaliana was expressed in Escherichia coli DH5α. 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 DH5α. 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 21oC and 3.6×10-3 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.
authors.affiliation: College of Life Science, Dezhou University Key University Laboratory of Biotechnology and Utilization of Bio-resource of Shandong Dezhou China manuscript_sub@126.com.
authors.forename: Q
authors.initials: Q
authors.lastname: He
authors.affiliation: College of Life Science, Dezhou University Key University Laboratory of Biotechnology and Utilization of Bio-resource of Shandong Dezhou China aiyun_fu@126.com.
authors.forename: A Y
authors.initials: AY
authors.lastname: Fu
authors.affiliation: College of Life Science, Dezhou University Key University Laboratory of Biotechnology and Utilization of Bio-resource of Shandong Dezhou China.
authors.forename: G C
authors.initials: GC
authors.lastname: Zhang
authors.affiliation: College of Life Science, Dezhou University Key University Laboratory of Biotechnology and Utilization of Bio-resource of Shandong Dezhou China.
authors.forename: T J
authors.initials: TJ
authors.lastname: Li
authors.affiliation: College of Life Science, Dezhou University Key University Laboratory of Biotechnology and Utilization of Bio-resource of Shandong Dezhou China.
authors.forename: J H
authors.initials: JH
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012639:Seeds / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D018398:Homeodomain Proteins; D011994:Recombinant Proteins; C121158:SEP3 protein, Arabidopsis; D014157:Transcription Factors