Simple purification of a foreign protein using polyhedrin fusion in a baculovirus expression system.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures, and that these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with the Polh gene at the N-terminus, including a linker and enterokinase (EK) site between Polh and EGFP, was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells in three steps: cell harvest, sonication, and EK digestion. Through final enterokinase digestion, EGFP presented mainly in the supernatant, and this supernatant fraction also showed a pure EGFP band. These results suggest that a combined procedure of Polh fusion expression and enterokinase digestion can be used for rapid and easy purification of other proteins.

Improved baculovirus vectors expressing barnase using promoters from Cotesia plutellae bracovirus.

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.

Identification and molecular characterization of novel cry1-type toxin genes from Bacillus thuringiensis K1 isolated in Korea.

To clone novel cry1-type genes from the Bacillus thuringiensis K1 isolate, about 2.4-kb-long PCR fragments were amplified with two primer sets of ATG1-F/N400-R and 1BeATG1-F/N400-R. Using PCR-RFLP, three novel cry1-type genes, cry1-1, cry1-7, and cry1-44, were obtained from B. thuringiensis K1 and the complete coding sequences of these novel genes were analyzed. The Cry1-1, Cry1-7, and Cry1-44 proteins showed maximum similarities of about 78.0%, 99.7%, and 91.0% with the Cry1Ha1, Cry1Be1, and Cry1Ac2 proteins, respectively. These novel cry1-type genes were expressed using a baculovirus expression vector system and their insecticidal activities were investigated. Whereas all three novel genes were toxic to Plutella xylostella larvae, only Cry1-1 showed insecticidal activity against Spodoptera exigua larvae.

Genomic segments cloning and analysis of Cotesia plutellae polydnavirus using plasmid capture system.

Cotesia plutellae polydnaviruses (CpBV) has a segmented genome consisting of multiple circular double stranded DNAs. Recently, we have developed an easy, simple, and convenient system based on Tn7 transposition in order to clone genomic segments of CpBV in Escherichia coli cell and designated plasmid capture system (PCS). The PCS donor-S transferred a pUC19 origin of replication and an ampicillin resistance marker into CpBV genomic DNA by in vitro transposition. Through PCS system, we were able to clone 53 genomic clones ranging from 0.1 to 25.5 kb and further they were classified into 29 segments by their sizes and restriction endonuclease patterns. Among them, a complete nucleotide sequence of CpBV-S28 segment was determined and 10 putative genes were predicted from this segment. Interestingly, 9 of 10 putative ORFs had high level of similarities with catalytic domain of protein tyrosine phosphatase. Also, ORF2807 showed similarity with EP1-like proteins of C. congregata polydnavirus.