The baculovirus expression vector system (BEVS) is an effective and widely used system for the production of recombinantproteins in insect cells or larvae. However, the expression efficiency of recombinant proteins using the polyhedrin promotercould not acquire the protein yields observed for native polyhedrin. In this study, we tried to develop hyper expressionvector by the optimal combination of previously reported various enhancer factors. The selected enhancer factors for optimalexpression consists homologous region5 (hr5), VP39 promoter and burst sequences. Seven recombinant viruses were madeto compare EGFP expression level. Each recombinant viruses showed different expression levels respectively, and themost of expression level was observed with higher than those of the previous vectors. This study suggests a new optionfor hyper expression of useful recombinant protein using the BEVS.

Viral particles of Porcine epidemic diarrhea virus (PEDV) consist of a four structural proteins. Among them Spikeprotein mediated responsible for receptor binding and membrane fusion during viral infection and therefore the main targetof neutralizing antibodies. Virus-like particles (VLPs) are consisted of one or more viral structural proteins, and theirmorphologies closely resemble those of the native virus. VLPs have no virulence and can elicit robust immune responsesas compared with inactivated or live-attenuated virus vaccines. Thus, in this study, we tried two methods for VLP constructionin Bombyx mori, one is traditional method and the other is chimeric VLP method using the influenza matrix protein.Both methods could produce successfully PEDV VLPs.

The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various enhancer factor were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). As a hyper expression factor, the optimal hyper BEVS was constructed by combining Hr5 sequence, VP39 promoter and Burst sequences. Additionally, the proteins expressed by the hyper expression system was markedly increased. This study suggests a new option for higher expression of useful foreign recombinant protein using the BEVS.

Recombinant proteins including a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. However, the affinity tag must be removed from the target after the purification process. Recently, the Synechocystis sp. PCC6803 DnaB mini-intein (Ssp DnaB mini-intein) is widely used in Escherichia coli expression systems as the solution of this problem. The Ssp DnaB mini-intein can be induced simply by shifting of pH and temperature, offering a benefit to cleave a peptide bond without using a protease or chemical reagent. Although the utility of this novel tag is widely studied in E. coli, there is no report yet in baculovirus expression vector system (BEVS). In this study, we generated several recombinant baculoviruses to express foreign proteins with Ssp DnaB mini-intein. In conclusion Ssp DnaB mini-intein was good tag also in BEVS with more advantages.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to meet a multi-parellel process. We have developed a novel recombinant bacmid, bEasyBm that enabling easy and fast generation of pure recombinant virus without any purification step. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus early promoter. Therefore, only when the barnase gene was replaced to gene of interest, the bEasyBm could replicate in host insect cells. When the bEasyBm was transposed with pDualBac-EGFP and pDualBac-LUC respectively, there were no non-recombinant backgrounds were detected from unpurified BmEasy-EGFP or BmEasy-LUC stocks. In addition, the resulting recombinant virus, BmEasy-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, BmEGFP, which was constructed using bBmGOZA system. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established.

Recently, we constructed a novel recombinant baculovirus genome, bEasyBac, enabling easy and fast generation of pure recombinant baculovirus without any purification step. In the bEasyBac, bacteriophage lambda site-specific attachment (att) sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus (CpBV) ORF3005 early promoter to negatively select against non-recombinant background. The bEasyBac could replicate in host insect cells only when the barnase gene was replaced to gene of interest by in vitro transposition. When the bEasyBac was transposed with pDualBac-EGFP and the EGFP expression efficiency along passage was investigated, the resulting recombinant virus, EasyBac-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, AcEGFP, which was constructed using bAcGOZA system, whereas, the non-purified AcEGFP showed quite reduced level of EGFP along passages. Moreover, no non-recombinant backgrounds were detected from unpurified EasyBac-EGFP stocks. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established. These results suggest that the bEasyBac has an effective benefit enabling for high-throughput baculovirus expression vector without purifying recombinant virus.

The flavin-containing monooxygenases (FMOs) (EC 1.14.13.8) are NADPH-dependent flavoenzymes that catalyze oxidation of soft nucleophilic heteroatom centers in a range of structurally diverse compounds including foods, drugs, pesticides, and other xenobiotics. In humans, FMO1 appears to be the predominant form expressed in human fetal liver. cDNA-expressed human FMO and human liver microsomal FMO have been observed to N- and S-oxygenate nucleophilic nitrogen- and sulfur-containing drugs and chemicals, respectively. In the present study, FMOl can be expressed in the baculovirus expression vector system at level of 2.68 nmol FMO1/㎎ of membrane protein. This isoform was examined for its capacity to metabolize methimazole to its S-oxide using thiocholine assay. Kinetic studies of its S-oxide by recombinant human FMO1 result in K_m of 7.66 μM and V_(max) of 17.79 nmol/min/㎎ protein.