Purification Of Recombinant Peritrophic Membrane Proteins Of The Old World Screwworm Fly, Chrysomya Bezziana / Pearson, Roger ; Muharsini, Sri[Research Institute for Veterinary Science]Wijffels, Gene ; Vuocolo, Tony

Jurnal Ilmu Ternak dan VeterinerEdisi Khusus .-- Vol.5 (3): p.185-191.-- ABSTRAK : 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 / Untuk mengevalusi kelayakan vaksinasi domba terhadap lalat the Old World Screwworm Chrysomya bezziana, beberapa protein peritrofin rekombinan telah diekspresikan baik pada Pichia pastoris dalam keadaan denaturasi maupun pada Escherichia coli dalam bentuk natif. Pemurnian protein dengan label hexaHis tersebut dilakukan dengan immobilized metal affinity chromatography. Protein yang dimurnikan dalam keadaan reduksi dinetralkan kembali dengan glutation. Pemurnian protein fusi S-transferase glutation dilakukan dengan menggunakan glutathione affinity chromatography dilanjutkan dengan anion exchange chromatography. Keaslian dari protein yang diekskresikan dievaluasi kembali dengan teknik perunutan asam amino dari asam amino terminal.Analisis karbohidrat dengan biotinylated lectin menunjukkan bahwa Cb-peritrophin-48 yang diekspresikan pada Pichia pastoris mengalami proses glikosilasi dengan gugus gula manosa dengan jumlah banyak. Empat protein rekombinan yang telah dimurnikan digunakan untuk mengevaluasi tingkat imunogenisitas protektifnya pada domba terhadap investasi larva lalat Chrysomya bezziana.