Molecular chaperone-assisted production of human alpha-1,4-N-acetylglucosaminyltransferase in silkworm larvae using recombinant BmNPV bacmids.

Abstract:

:In this study, human alpha-1,4-N-acetylglucosaminyltransferase (alpha4GnT) fused with GFP(uv) (GFP(uv)-alpha4GnT) was expressed using both a transformed cell system and silkworm larvae. A Tn-pXgp-GFP(uv)-alpha4GnT cell line, isolated after expression vector transfection, produced 106 mU/ml of alpha4GnT activity in suspension culture. When Bombyx mori nucleopolyhedrovirus containing a GFP(uv)-alpha4GnT fusion gene (BmNPV-CP (-)/GFP(uv)-alpha4GnT) bacmid was injected into silkworm larvae, alpha4GnT activity in larval hemolymph was 352 mU/ml, which was 3.3-fold higher than that of the Tn-pXgp-GFP(uv)-alpha4GnT cell line. With human calnexin (CNX) or human immunoglobulin heavy chain-binding protein (BiP, GRP78) coexpressed under the control of the ie-2 promoter, alpha4GnT activity in larval hemolymph increased by 1.4-2.0-fold. Moreover, when BmNPV-CP (-)/GFP(uv)-alpha4GnT bacmid injection was delayed for 3 h after BmNPV-CP (-)/CNX injection, the alpha4GnT activity increased significantly to 922 mU/ml, which was 8.7-fold higher than that of the Tn-pXgp-GFP(uv)-alpha4GnT cell line. Molecular chaperone assisted-expression in silkworm larvae using the BmNPV bacmid is a promising tool for recombinant protein production. This system could lead to large-scale production of more complex recombinant proteins.

journal_name

Mol Biotechnol

journal_title

Molecular biotechnology

authors

Nakajima M,Kato T,Kanamasa S,Park EY

doi

10.1007/s12033-009-9174-8

subject

Has Abstract

pub_date

2009-09-01 00:00:00

pages

67-75

issue

1

eissn

1073-6085

issn

1559-0305

journal_volume

43

pub_type

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