Expression, purification and characterization of recombinant PLCζ protein in baculovirus-insect cell expression system.
- Author:
Xin CHEN
1
;
Yueyue HU
1
;
Hongyi XU
1
;
Xiaoyan WANG
2
;
Kai DENG
1
Author Information
- Publication Type:Journal Article
- Keywords: PLCζ protein; baculovirus expression system; expression; identification; insect cell; purification
- MeSH: Animals; Baculoviridae; Genetic Vectors; Humans; Recombinant Proteins; Sf9 Cells; Spodoptera
- From: Chinese Journal of Biotechnology 2019;35(6):1135-1142
- CountryChina
- Language:Chinese
- Abstract: PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
