A stable Tn-5B1-4 insect cell line co-expressing the recombinant GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 (GFPuv-beta3GnT2)
protein fused to a
melittin signal sequence with a
lectin-like
molecular chaperone, human
calnexin (hCNX) or human
calreticulin (hCRT), was constructed. The expression of either of these
molecular chaperones is under the control of a weak promoter, OpMNPV IE2, while that of GFPuv-beta3GnT2 is under the control of Bombyx mori actin promoter. This co-expression system was compared between two different insect cell-baculovirus expression systems: (1)
co-infection of the recombinant baculovirus containing a
molecular chaperone (AcNPV-hCNX or -hCRT) with a recombinant baculovirus containing GFPuv-beta3GnT2 fused with the
melittin signal sequence (AcNPV-me-GGT); (2)
infection of AcNPV-me-GGT to a stably expressing cell line for either hCNX or hCRT. In the
co-infection system, the intracellular GFPuv-beta3GnT2 expression level was low because of the improved secretion level ratio of the fusion
protein, due to the chaperone expression. In the case of
infection to the stably expressing cell line for a chaperone, the extracellular GFPuv-beta3GnT2 expression level was similar to the intracellular expression level. This suggests that the amount of expressed chaperone is not sufficient to process beta3GnT2. On the other hand, the co-expression system produced an extracellular beta3GnT activity of 22-23 mU/mL, which was approximately 3.5- and 11-fold higher than those of the stable expression of the fusion gene without the chaperone and the conventional
BES with the addition of
protease, respectively. The secretion level ratio of the fusion
protein of this system increased to 82%, which was approximately 1.5-fold that of any other expression system investigated thus far. These results indicate that the ratio of the expression level of the target gene to that of the chaperone gene may be an important factor in maximizing the production of a target
protein. The
molecular-chaperone-assisted expression system using a stably transformed insect cell line offers promising prospects for the efficient production of recombinant secretory
proteins in insect cells.