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Expression and purification of a recombinant Fip‐fve protein from Flammulina velutipes in baculovirus‐infected insect cells
Author(s) -
Wu C.M.,
Wu T.Y.,
Kao S.S.,
Ko J.L.,
Jinn T.R.
Publication year - 2008
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2007.03686.x
Subject(s) - biology , recombinant dna , signal peptide , escherichia coli , western blot , microbiology and biotechnology , biochemistry , gene
Aims: To develop an efficient and facile expression system supply of high purity and stable activity of rFip‐fve for oral administration, medicinal study and applications. Methods and Results: A recombinant virus that contained the chimera gene, encoding a bombyxin signal peptide sequence fused to a Fip‐fve‐6His sequence, was constructed. The rFip‐fve was purified from the supernatant of the infected Sf21 cells using a nickel‐chelated affinity column, and was verified by Western blot and MALDI‐MS (matrix‐assisted laser desorption ionization mass spectrometry) analyses. Results showed that a glycosylated mature rFip‐fve was produced and secreted into the infected cell supernatant. The immunomodulatory activity of rFip‐fve was evaluated by measuring the amount of interleukin‐2 released from murine splenocytes. Conclusions: A reliable scheme to express and purify active rFip‐fve in a baculovirus/insect cell system for medicinal applications and genetic study is a feasible means of solving potential problems related to the production and activity of rFip‐fve protein. Significance and Impact of the Study: The rFip‐fve expressed in insect cells was processed and modified in a manner more similar to that of its native counterpart than that in bacterial cells. Therefore, the potential applications of rFip‐fve that is generated in Sf21 cells can be more effectively evaluated that produced in Escherichia coli .