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Improvement of the production of GFP uv ‐β1,3‐ N ‐acetylglucosaminyltransferase 2 fusion protein using a molecular chaperone‐assisted insect‐cell‐based expression system
Author(s) -
Kato Tatsuya,
Murata Takeomi,
Usui Taichi,
Park Enoch Y.
Publication year - 2004
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20362
Subject(s) - green fluorescent protein , chaperone (clinical) , fusion protein , biology , microbiology and biotechnology , heat shock protein , recombinant dna , extracellular , signal peptide , gene , biochemistry , medicine , pathology
A stable Tn‐5B1‐4 insect cell line co‐expressing the recombinant GFP uv ‐β1,3‐ N ‐acetylglucosaminyltransferase 2 (GFP uv ‐β3GnT2) 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 GFP uv ‐β3GnT2 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 GFP uv ‐β3GnT2 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 GFP uv ‐β3GnT2 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 GFP uv ‐β3GnT2 expression level was similar to the intracellular expression level. This suggests that the amount of expressed chaperone is not sufficient to process β3GnT2. On the other hand, the co‐expression system produced an extracellular β3GnT 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. © 2004 Wiley Periodicals, Inc.