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Updated: Mar 29, 2026
GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
Published on: October 29, 2013
[Expression and affinity purification of Mycobacterium tuberculosis His-LprG-FLAG in HEK293T cells]
Bing Bai1, Min Su2, Minyan Liu1
1Department of Medicine Laboratory, Guangxi Medical University, Department of Clinical Laboratory, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
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.
Conclusion:
Fusion protein His-LprG-FLAG has been successfully prepared and expressed in HEK293T cells.
