title: [Expression and affinity purification of Mycobacterium tuberculosis His-LprG-FLAG in HEK293T cells].
abstract: OBJECTIVE
  To construct a eukaryotic expressing vector of Mycobacterium tuberculosis lipoprotein G (LprG), and express and purify the recombinant protein in HEK293T cells by affinity chromatography.
  
  
  METHODS
  The LprG gene was amplified by PCR from the genome of MTB strain H37Rv. The subsequent PCR product and eukaryotic expressing vector pcDNA3.1 were digested by certain restriction enzymes. The recombinant vector, pcDNA3.1-His-LprG-FLAG, was constructed by ligation of target genes and the vector. After identified by enzymes digestion and DNA sequencing analysis, the correct recombinant vector was applied for transfection of HEK293T cells. The expression of His-LprG-FLAG was examined by Western blotting. The target fusion protein successfully expressed in HEK293T cells was purified by Ni(2+) affinity chromatography, and the purification and concentration of the protein was evaluated by SDS-PAGE and Western blotting.
  
  
  RESULTS
  The recombinant vector pcDNA3.1-His-LprG-FLAG was successfully constructed, which was identified by double enzyme digestion and DNA sequencing. Western blotting indicated that the fusion protein was expressed in HEK293T cells transfected with the vector, with the molecular mass (Mr) being 27 000. By Ni2+ affinity chromatography, the fusion protein could be purified to a high concentration, as evaluated by SDS-PAGE and Western blotting.
  
  
  CONCLUSIONS
  Fusion protein His-LprG-FLAG has been successfully prepared and expressed in HEK293T cells.
authors.affiliation: Department of Medicine Laboratory, Guangxi Medical University, Department of Clinical Laboratory, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Bai
authors.affiliation: Department of Medicine Laboratory, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.affiliation: Department of Medicine Laboratory, Guangxi Medical University, Department of Clinical Laboratory, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
authors.forename: Minyan
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Medicine Laboratory, Guangxi Medical University, Department of Clinical Laboratory, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
authors.forename: Taijie
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Medicine Laboratory, Guangxi Medical University, Department of Clinical Laboratory, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Medicine Laboratory, Guangxi Medical University, Department of Clinical Laboratory, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China. *Corresponding authors, E-mail: lianghongjie2004@163.com.
authors.forename: Hongjie
authors.initials: H
authors.lastname: Liang
authors.affiliation: Shanghai Key Laboratory of Tuberculosis, Tuberculosis Diagnosis and Treatment Center, Shanghai Pulmonary Hospital, Tongji University, Shanghai 200433, China. *Corresponding authors, E-mail: chenjindor@126.com.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins