Biotechnol. Appl. Biochem. (2008) 49, 135–140 (Printed in Great Britain) doi:10.1042/BA20070098 135 Improved expression of fusion protein using a cysteine- protease- and chitinase-deficient Bombyx mori (silkworm) multiple nucleopolyhedrovirus bacmid in silkworm larvae Enoch Y. Park*† 1 , Takahiro Abe† and Tatsuya Kato† *Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan, and †Laboratory of Biotechnology, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan A bacmid system of BmMNPV [Bombyx mori (silkworm) multiple nucleopolyhedrovirus] with both the cysteine-protease and chitinase genes deleted (BmMNPV-CP - -Chi - ) was constructed. The viral protease and chitinase activities in the haemolymph of Bombyx mori larvae infected with this BmMNPV- CP - -Chi - bacmid were reduced by 95 and 50 % respectively. By using this system, a GFP uv –β 3GnT2 (green fluorescent protein excited by UV light–β 1,3- N-acetylglucosaminyltransferase 2) fusion protein was successfully expressed in silkworm larvae with less protein degradation and without larvae liquefaction; β 3GnT2 activity improved 2.8-fold over that of unmodified bacmid. This BmMNPV-CP - -Chi - bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation. Introduction The baculovirus expression system is widely used and often expresses recombinant proteins at high levels in cultured insect cells, as its expression is driven by the strong polyhedrin promoter. It also has great advantages for expression of eukaryotic proteins, since the insect cells can execute the post-translational modifications into proteins similar to their native types [1–3] in a manner that bacterial expression systems cannot. AcMNPV [Autographa californica (alfalfa looper) multiple nucleopolyhedrovirus] or BmMNPV [Bombyx mori (silkworm) multiple nucleopolyhedrovirus] have been used for protein expression in insect cells. AcMNPV can only be used in cell lines from Spodoptera frugiperda (fall armyworm), Trichoplusia ni (cabbage looper) and their larvae. On the other hand, BmMNPV can be used in both silkworm larva and its cell line. These BmMNPV vectors are suitable for the large-scale production of recombinant proteins, resulting in improvements of protein yield [4]. Recently, Motohashi et al. [5] have developed the first practical BmMNPV bacmid system directly applicable for protein expression in silkworm larvae. This bacmid system can express the proteins much more rapidly than the system which was previously used, since the bacmid can be replicated in Escherichia coli as a large plasmid and generate the recombinant virus DNA by the site-specific transposition in E. coli and remain infectious to insect cells. This method provides the rapid protein production in silkworm for a period as long as 10 days, and is free from biohazards, because there is no need to use a baculovirus for infection. This bacmid will be a powerful tool for the future production of recombinant eukaryotic proteins, because the bacmid does not require any baculovirus amplification step. However, the silkworm larvae were liquefied at around 5 days post-infection. Host liquefaction is caused by a papain- type cysteine protease with cathepsin L-like characteristics, and a chitinase, ChiA [6–9]. For production of recombinant proteins in silkworm larvae, host liquefaction often leads to the loss of proteins. Suzuki et al. [10] constructed a BmMNPV lacking the cysteine-protease gene and produced firefly luciferase and human growth factor in the silkworm very efficiently. This was due to the markedly reduced degradation that is usually caused by the cysteine protease. In the present study, a BmMNPV-CP − -Chi − (cysteine- protease- and chitinase-deficient BmMNPV) bacmid was constructed using the lambda recombination system. Key words: bacmid, Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), chitinase, cysteine protease, green fluorescent protein excited with long-wave UV light–β1,3-N-acetylglucosaminyltransferase 2 (GFP uv –β3GnT2) fusion protein, silkworm (Bombyx mori). Abbreviations used: AcMNPV, Autographa californica (alfalfa looper) multiple nucleopolyhedrovirus; bacmid, baculovirus shuttle vector; BmMNPV, Bombyx mori multiple nucleopolyhedrovirus; BmMNPV-CP − -Chi − , cysteine-protease- and chitinase-deficient BmMNPV; CAT, chloramphenicol acetyltransferase; DMRE-C reagent, N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide; GFP uv , green fluorescent protein when excited with long-wave UV light; GGT2, GFP uv –β3GnT2 (β1,3-N-acetylglucosaminyltransferase 2) fusion protein; IPTG, isopropyl β-D-thiogalactopyranoside; me-GGT2, GGT2 gene fused with the melittin signal sequence; X-Gal, 5-bromo-4-chloroindol-3-yl β-D-galactopyranoside. 1 To whom correspondence should be addressed (email yspark@agr.shizuoka.ac.jp). C  2008 Portland Press Ltd