Joong Nam Kang

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.

Dual Insecticidal Activity of Spodoptera-Toxic Bacillus thuringiensis Strain Transformed with Lepidopteran-Specific Cry Toxin

Abstract The E. coli-B. thuringiensis shuttle vector for expression of cry1Ac , pHT1K-1Ac plasmid was introduced into acrystalliferous B. thuringiensis Cry − B and Spodoptera toxic STB-3 strain. The presence of a recombinant plasmid in transformants after electroporation was confirmed by PCR. The 1K-1Ac/Cry − B(Cry − B transformant) and 1K-1Ac/STB-3 (STB-3 transformant) produced bipyramidal-shaped parasporal inclusion that was 130 kDa in size as like B. thuringiensis subsp. kurstaki HD-73. In P. xylostella bioassay, these transformants showed significantly high toxicity than the wild-type recipients and further, in case of B. thuringiensis STB-3 transformant still had original Spodoptera toxicity. These results suggested that the pHT1K could be successfully applied for generating individual B. thuringiensis strains that produce various combinations of insecticidal proteins to expand their host spectrum and enhance insecticidal activity.

Identification of a Novel Crystal Protein Gene from Bacillus thuringiensis Isolated in Korea

To identify novel cry1-type crystal protein genes, 100 Bacillus thuringiensis (Bt) isolates were selected on the basis of their toxicity against lepi- dopteran insect larvae. For rapid search of a large quantity of novel genes simultaneously, Bt isolates were randomly divided into 2 groups including 50 isolates each. About 2.4 kb PCR fragments were amplified from each group using universal oligonu- cleotide primers, ATG1-F and N400-R, designed to probe the toxic fragment regions of all known and possible cry1-type genes. Restriction fragment length polymorphism (RFLP) analysis revealed 3 distinct patterns in each group. One of them, A32 clone showed 87% and 91% of nucleotide sequence and deduced amino acid sequence similarities with the cry1Ea gene. Interestingly, nucleotide sequence ana- lysis of A32 cry gene suggested that this gene might have resulted from nucleotide rearrangement between cry1Ac and cry1Ea genes.