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Bacterial expression of larval peritrophins of Chryosomya bezziana Wijffels, Gene; Voucoloco, Tony; Muharsini, Sri; Supriyanti, Florentina
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 3 (2000)
Publisher : Indonesian Animal Sciences Society

Three candidate antigens, Chrysomya bezziana peritrophin-48, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42, were prepared for recombinant protein production in Escherichia coli using a variety of expression vectors. Cb peritrophin-48 was expressed as a recombinant protein possessing a carboxy-terminal hexaHis tag. Cb peritrophin-15 was expressed as both a glutathione S-transferase fusion protein and as an amino-terminal hexaHis tagged protein. The glutathione Stransferase Cb peritrophin-15 construct produced a heterogeneous group of fusion proteins. Cb peritrophin-42 was also expressed as an amino-terminal hexaHis tagged protein. The two putative domains of Cb peritrophin-42 were also separately expressed, again with amino-terminal hexaHis tags. Cultures of the hexaHis constructs Cb peritrophin-48, -15 and â42 were demonstrated to be useful for the production and purification of these protein antigens and were scaled-up for vaccine trials and protein characterization studies. Â Key words: Chrysomya, peritrophic membrane, peritrophin, recombinant protein, hexaHis tag, GST

Expression in yeast (Pichia pastoris) of recombinant Cb-peritrophin-42 and Cbperitrophin- 48 isolated from Chrysomya bezziana (the Old World Screwworm fly) Muharsini, Sri; Voucoloco, Tony
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 3 (2000)
Publisher : Indonesian Animal Sciences Society

Pichia pastoris has been investigated as a means to express recombinant forms of two putative peritrophic membrane antigens from Chrysomya bezziana, Cb-peritrophin-42 (Cb42) and Cb-peritrophin-48 (Cb48). Recombinant Cb48 was expressed as a secreted and glycosylated protein. The yield of recombinant protein was 8 mg per litre of culture. In contrast, recombinant Cb42 was not expressed at detectable levels in Pichia pastoris, probably due to A + T rich sequence which may cause premature transcriptional termination. To expedite Cb42 expression in yeast, Cb42 was divided into two domains: Cb42A and Cb42B. Cb42B was successfully expressed in Pichia pastoris, yielding 0.4 mg per litre of culture. However, Cb42A was not expressed. This work demonstrates that although Pichia pastoris offers considerable benefits as an expression system producing high level of glycosylated protein, success may vary from protein to protein. Â Key words: Chrysomya bezziana, peritrophin, Pichia pastoris

Purification of recombinant peritrophic membrane proteins of the Old World Screwworm fly, Chrysomya bezziana Pearson, Roger; Muharsini, Sri; Wijffels, Gene; Voucoloco, Tony
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 3 (2000)
Publisher : Indonesian Animal Sciences Society

To evaluate the feasibility of vaccinating sheep against the Old World Screwworm fly Chrysomya bezziana several recombinant peritrophin proteins were expressed in either a denatured form in Escherichia coli or a native-like form in Pichia pastoris cultures. Purification of the hexaHis tagged proteins was achieved by immobilized metal affinity chromatography. Proteins purified under reducing conditions were refolded using a glutathione shuffle procedure. Purification of a glutathione-Stransferase fusion protein was attempted using glutathione affinity chromatography in conjunction with anion exchange chromatography. The authenticity of the expressed proteins was verified by amino terminal amino acid sequencing. Carbohydrate analysis using biotinylated lectins revealed that Cb-peritrophin-48 expressed in Pichia pastoris was glycosylated with high mannose-type sugars. Four of the purified recombinant proteins were used to evaluate their protective immunogenicity in sheep against Chrysomya bezziana strike. Â Key words: Screwworm fly, Chrysomya bezziana, recombinant proteins, immobilized metal affinity chromatography

Vaccination trials in sheep against Chrysomya bezziana larvae using the recombinant peritrophin antigens Cb15, Cb42 and Cb48 ., Sukarsih; Partoutomo, Sujitno; Wijffels, Gene; Voucoloco, Tony; Willadsen, Peter
Indonesian Journal of Animal and Veterinary Sciences Vol 5, No 3 (2000)
Publisher : Indonesian Animal Sciences Society

Recombinant forms of a number of peritrophic membrane proteins from the screwworm fly Chrysomya bezziana have been assessed in vitro and in vivo for their efficacy as antigens in vaccination against the tissue-invasive, larval form of the parasite. The proteins included Cb15 and Cb42 expressed in Escherichia coli and Cb48 expressed in both Escherichia coli and Pichia pastoris. In all cases, the in vitro assays of larval growth on serum from vaccinated sheep failed to show inhibition of larval weight gain or any detrimental effect on larval survival relative to controls. Chrysomya bezziana Cb48 has a significant degree of sequence identity with the antigen PM48 from Lucilia cuprina. Feeding Lucilia cuprina larvae on antisera to Cb48 induced a small but statistically significant reduction in weight gain, as does feeding on antisera to PM48. In vivo, larvae feeding on sheep vaccinated with Escherichia coli-expressed Cb15 and Cb42 and Pichia pastoris-expressed Cb48 showed marginally greater weight gain and survival which was equal to or greater than that on non-vaccinated sheep. The significance of these observations is discussed. Â Key words: Chrysomya bezziana, recombinant antigen, peritrophin, vaccination

Vaccination trials in sheep against Chrysomya bezziana larvae using the recombinant peritrophin antigens Cb15, Cb42 and Cb48 Sukarsih .; Sujitno Partoutomo; Gene Wijffels; Tony Voucoloco; Peter Willadsen
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Recombinant forms of a number of peritrophic membrane proteins from the screwworm fly Chrysomya bezziana have been assessed in vitro and in vivo for their efficacy as antigens in vaccination against the tissue-invasive, larval form of the parasite. The proteins included Cb15 and Cb42 expressed in Escherichia coli and Cb48 expressed in both Escherichia coli and Pichia pastoris. In all cases, the in vitro assays of larval growth on serum from vaccinated sheep failed to show inhibition of larval weight gain or any detrimental effect on larval survival relative to controls. Chrysomya bezziana Cb48 has a significant degree of sequence identity with the antigen PM48 from Lucilia cuprina. Feeding Lucilia cuprina larvae on antisera to Cb48 induced a small but statistically significant reduction in weight gain, as does feeding on antisera to PM48. In vivo, larvae feeding on sheep vaccinated with Escherichia coli-expressed Cb15 and Cb42 and Pichia pastoris-expressed Cb48 showed marginally greater weight gain and survival which was equal to or greater than that on non-vaccinated sheep. The significance of these observations is discussed. Key words: Chrysomya bezziana, recombinant antigen, peritrophin, vaccination

Bacterial expression of larval peritrophins of Chryosomya bezziana Gene Wijffels; Tony Voucoloco; Sri Muharsini; Florentina Supriyanti
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Three candidate antigens, Chrysomya bezziana peritrophin-48, Chrysomya bezziana peritrophin-15 and Chrysomya bezziana peritrophin-42, were prepared for recombinant protein production in Escherichia coli using a variety of expression vectors. Cb peritrophin-48 was expressed as a recombinant protein possessing a carboxy-terminal hexaHis tag. Cb peritrophin-15 was expressed as both a glutathione S-transferase fusion protein and as an amino-terminal hexaHis tagged protein. The glutathione Stransferase Cb peritrophin-15 construct produced a heterogeneous group of fusion proteins. Cb peritrophin-42 was also expressed as an amino-terminal hexaHis tagged protein. The two putative domains of Cb peritrophin-42 were also separately expressed, again with amino-terminal hexaHis tags. Cultures of the hexaHis constructs Cb peritrophin-48, -15 and –42 were demonstrated to be useful for the production and purification of these protein antigens and were scaled-up for vaccine trials and protein characterization studies. Key words: Chrysomya, peritrophic membrane, peritrophin, recombinant protein, hexaHis tag, GST

Expression in yeast (Pichia pastoris) of recombinant Cb-peritrophin-42 and Cbperitrophin- 48 isolated from Chrysomya bezziana (the Old World Screwworm fly) Sri Muharsini; Tony Voucoloco
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Pichia pastoris has been investigated as a means to express recombinant forms of two putative peritrophic membrane antigens from Chrysomya bezziana, Cb-peritrophin-42 (Cb42) and Cb-peritrophin-48 (Cb48). Recombinant Cb48 was expressed as a secreted and glycosylated protein. The yield of recombinant protein was 8 mg per litre of culture. In contrast, recombinant Cb42 was not expressed at detectable levels in Pichia pastoris, probably due to A + T rich sequence which may cause premature transcriptional termination. To expedite Cb42 expression in yeast, Cb42 was divided into two domains: Cb42A and Cb42B. Cb42B was successfully expressed in Pichia pastoris, yielding 0.4 mg per litre of culture. However, Cb42A was not expressed. This work demonstrates that although Pichia pastoris offers considerable benefits as an expression system producing high level of glycosylated protein, success may vary from protein to protein. Key words: Chrysomya bezziana, peritrophin, Pichia pastoris

Purification of recombinant peritrophic membrane proteins of the Old World Screwworm fly, Chrysomya bezziana Roger Pearson; Sri Muharsini; Gene Wijffels; Tony Voucoloco
Jurnal Ilmu Ternak dan Veteriner Vol 5, No 3 (2000): SEPTEMBER 2000
Publisher : Indonesian Center for Animal Research and Development (ICARD)

To evaluate the feasibility of vaccinating sheep against the Old World Screwworm fly Chrysomya bezziana several recombinant peritrophin proteins were expressed in either a denatured form in Escherichia coli or a native-like form in Pichia pastoris cultures. Purification of the hexaHis tagged proteins was achieved by immobilized metal affinity chromatography. Proteins purified under reducing conditions were refolded using a glutathione shuffle procedure. Purification of a glutathione-Stransferase fusion protein was attempted using glutathione affinity chromatography in conjunction with anion exchange chromatography. The authenticity of the expressed proteins was verified by amino terminal amino acid sequencing. Carbohydrate analysis using biotinylated lectins revealed that Cb-peritrophin-48 expressed in Pichia pastoris was glycosylated with high mannose-type sugars. Four of the purified recombinant proteins were used to evaluate their protective immunogenicity in sheep against Chrysomya bezziana strike. Key words: Screwworm fly, Chrysomya bezziana, recombinant proteins, immobilized metal affinity chromatography

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