Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of <Emphasis Type="Italic">Penaeus monodon</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the -factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.     

Specific mutations within the alpha4-alpha5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry delta-endotoxins suggests that helices alpha4 and alpha5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the alpha4-alpha5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-beta-Dthiogalactopyranoside induction. When E. coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity. Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the alpha4-alpha5 loop play a crucial role in the larvicidal activity of the Cry4B toxin.     

High intracellular expression of giant catfish growth hormone under the control of PGK promoter in Saccharomyces cerevisiae

Different yeast plasmid systems containing different promoters such as ADH1, PGK, GAPDH and GAL1, and different selectable markers, such as URA3, TRP1 and leu2-d were compared to obtain the yeast expression system that provides high intracellular expression of giant catfish growth hormone (gcGH). The highest level of gcGH expression was observed in a recombinant yeast under the control of PGK promoter (17.1 mg/l or 1.4 g/0.1 OD). The amount of gcGH was increased six-fold (102.5 mg/l) when cells were grown in a rich medium (YEPD) with the inoculum and medium ratio of 1:1, although the amount of gcGH expression per cell density did not increase (1.0 g/0.1 OD). This indicated that the increased yield of gcGH in rich medium was due to the increased cell density. The aim of the study was to produce high level gcGH in the cells of S. cerevisiae in order to use the yeast cells as potential feed additives to promote growth in giant catfish.     

Development of a method for heterologous gene expression in Enterobacter amnigenus, a potential host for the biological control of mosquito larvi

An integrative plasmid containing a 1.3 kb fragment of chromosomal DNA from Enterobacter amnigenus was constructed. The Omega fragment encoding spectinomycin/streptomycin resistance was cloned into the unique BglII site of the resulting plasmid, and the interrupted fragment was transferred via plasmid pMAK705 by electroporation into E. amnigenus with a selection for spectinomycin resistance. Cointegrants were resolved to generate an E. amnigenus strain that expressed spectinomycin resistance, but grew as rapidly as the parental strain. The cloned fragment encodes a putative homologue of the proW gene of Escherichia coli that is not essential for E. amnigenus growth. The integrative plasmid is now available to introduce any heterologous DNA into the E. amnigenus chromosome, for the construction of promoter-probe vectors for the studies of gene regulation, or to construct plasmids suitable for the isolation of secretion signals. Immediate applications of this system will include the expression and secretion of crystal toxins from bacilli for the biological control of mosquito larvae infected with the bacterial host.     

Cloning and Characterization of a Mosquito Larvicidal Toxin Produced During Vegetative Stage of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> 2297

The mosquitocidal toxin 1 (mtx1) gene from genomic DNA of B. sphaericus strain 2297 was cloned and expressed in E. coli. DNA sequencing analysis of the cloned gene revealed a single open reading frame encoding an 870-amino acid polypeptide. Expression level of the full-length gene in E. coli was very low even though strong promoter was used or the gene was expressed as a fusion protein. Expression level was highly improved after the putative leader sequence was deleted, and the truncated gene was expressed as a fusion protein with glutathione S-transferase (GST-tMtx1). E. coli cells expressing GST-tMtx1 was highly toxic to Culex quinquefasciatus larvae and showed lower toxicity against Anopheles dirus and Aedes aegypti larvae. Enterobacter amnigenus An11, a mosquito larval gut colonizable bacteria, transformed with the cloned gene exhibited mosquito larvicidal activity. Result suggested that there is a potential to develop this protein to be used as an alternative mosquito control agent.     

Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae

Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.     

Aromaticity of Tyr-202 in the alpha4-alpha5 loop is essential for toxicity of the Bacillus thuringiensis Cry4A toxin

The current model for the mechanism of action of the Bacillus thuringiensis Cry delta-endotoxins involves the penetration of the alpha4-alpha5 hairpin into the target midgut epithelial cell membranes, followed by pore formation. In this study, PCR-based mutagenesis was employed to identify a critical residue within the alpha4-alpha5 loop of the 130kDa Cry4A mosquito-larvicidal protein. Alanine-substitutions of two charged (Asp-198 and Asp-200) and four polar (Asn-190, Asn-195, Tyr-201 and Tyr-202) residues in the alpha4-alpha5 loop were performed. Like the wild-type, all of the mutant toxins were over-expressed as inclusion bodies in Escherichia coli. When E. coli cells expressing each mutant toxin were bioassayed against Aedes aegypti larvae, larvicidal activity was completely abolished for the substitution of only Tyr-202, while replacements at the other positions still retained a high level of toxicity. Further replacement of Tyr-202 with an aromatic side chain, phenylalanine, did not affect the toxicity. These results revealed a crucial role in toxin activity for the conserved aromatic residue at the 202 position within the alpha4-alpha5 loop of the Cry4A toxin.