Expression analysis of a modified factor X in stably transformed insect cell lines.
A modified Factor X protein was combined with a cellulose-binding domain tag and expressed in insect cell lines. The protein, CBDFX, was expressed and secreted into the medium. Stable, transformed Hi5 and Sf9 insect cell lines were generated and tested for production of secreted CBDFX. The highest Sf9 and Hi5 CBDFX-producing cell lines were scaled up to 2-liter fermentors to evaluate production of this recombinant protein. Secreted protein production levels reached 4 mg/liter for the stable, transformed Hi5 cell line and 18 mg/liter for the stable, transformed Sf9 cell line. The protein was properly processed as determined by amino terminal sequencing and bound well to the cellulose substrate Avicel. In addition the activated recombinant CBDFX(a) was capable of recognizing and efficiently processing a Factor X cleavage site.
Pfeifer
T A
TA
Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. pfeifer@zoology.ubc.ca
Guarna
M M
MM
Kwan
E M
EM
Lesnicki
G
G
Theilmann
D A
DA
Grigliatti
T A
TA
Kilburn
D G
DG
D000818:Animals; D002461:Cell Line, Transformed; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D005170:Factor X / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D005822:Genetic Vectors; D007313:Insecta; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D005170:Factor X; D002482:Cellulose