Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Wu
C-M
CM
Biopesticide Department, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Wufeng, Taiwan.
Wu
T-Y
TY
Kao
S-S
SS
Ko
J-L
JL
Jinn
T-R
TR
D000818:Animals; D016367:Baculoviridae / Q000502:physiology*; D019149:Bioreactors; D002460:Cell Line; D002478:Cells, Cultured; D004926:Escherichia coli; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D007155:Immunologic Factors / Q000302:isolation & purification* / Q000494:pharmacology; D007218:Industrial Microbiology; D007376:Interleukin-2 / Q000096:biosynthesis; D037102:Lectins / Q000302:isolation & purification* / Q000494:pharmacology; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D013154:Spleen / Q000187:drug effects / Q000276:immunology; D018411:Spodoptera / Q000821:virology
D005656:Fungal Proteins; D007155:Immunologic Factors; D007376:Interleukin-2; D037102:Lectins; D011994:Recombinant Proteins