Expression and purification of an antitumor-analgesic peptide from the venom of Mesobuthus martensii Karsch by small ubiquitin-related modifier fusion in Escherichia coli
| Autor: | Min Wang, Zhigang Wang, Xueting Cai, Zhenhua Gu, Peng Cao, Wuguang Lu, Jiemiao Yu, Tingmei Ye, Juan Zhang |
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| Rok vydání: | 2010 |
| Předmět: |
medicine.medical_treatment
Mesobuthus martensii Recombinant Fusion Proteins SUMO-1 Protein Scorpion Venoms Peptide Antineoplastic Agents Protein aggregation Biology Chromatography Affinity law.invention Scorpions chemistry.chemical_compound Affinity chromatography law Cell Line Tumor medicine Escherichia coli Animals Humans chemistry.chemical_classification Analgesics Protease Coomassie Brilliant Blue biology.organism_classification Fusion protein Molecular biology chemistry Biochemistry Recombinant DNA Electrophoresis Polyacrylamide Gel Peptides Biotechnology |
| Zdroj: | Biotechnology progress. 26(5) |
| ISSN: | 1520-6033 |
| Popis: | To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the antitumor-analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO-AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni-NTA affinity chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N-terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 |
| Databáze: | OpenAIRE |
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