title: Protein expression and purification of human Zbtb7A in Pichia pastoris via gene codon optimization and synthesis.
abstract: Human Zbtb7A was proved to be an important molecular switch in oncogenesis. However, it is difficult to obtain its protein expression in prokaryotic system, due to high G+C content and rare codons in zbtb7a gene. Therefore, to further research the function and application of this protein, we optimized its coding sequence according to the codon bias of Pichia pastoris, synthesized the sequence with two-step PCR and confirmed the accuracy by DNA sequencing. The assembled fragment was introduced into P. pastoris expression vector pPIC9K and the resultant plasmid pPIC9K-zbtb7a-his(6) was transformed into the P. pastoris strain GS115 by electroporation. The products of the transformants induced by methanol were analyzed by 10% SDS-PAGE and identified by Western Blot assay. The expression conditions of the selected transformant were optimized. Additionally, a two-step purification protocol was applied to purify the recombinant protein. The results showed that the synthetic coding sequence of human Zbtb7A was successfully obtained and inserted into pPIC9K vector. Human Zbtb7A protein was expressed in P. pastoris and identified by western blot. The optimal conditions for its expression in P. pastoris were under a final concentration of 1% methanol and a time-course of 4d. Through the two-step purification, Zbtb7A protein was purified in high purity and its production reached up to as high as 18 mg/L. These results indicated that an effective procedure for expressing and purifying human Zbtb7A in P. pastoris was established.
authors.affiliation: Immunology and Reproduction Biology Lab, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Qingling
authors.initials: Q
authors.lastname: Wang
authors.forename: Feifan
authors.initials: F
authors.lastname: Zhang
authors.forename: Yahong
authors.initials: Y
authors.lastname: Huang
authors.forename: Yulian
authors.initials: Y
authors.lastname: Ji
authors.forename: Yayi
authors.initials: Y
authors.lastname: Hou
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003062:Codon; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D003062:Codon; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C485514:ZBTB7A protein, human