Expression, purification and characterization of recombinant human β-amyloid 1–42 in Pichia pastoris
| Autor: | Weiqun Yan, Xupeng Mu, Hao Xu, Mohan Shen, Quancai Wang |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Molecular Sequence Data
Gene Expression Peptide Biology Pichia Pichia pastoris law.invention Sepharose Bioreactors law Complementary DNA Gene expression Humans Amino Acid Sequence Cloning Molecular Peptide sequence chemistry.chemical_classification Amyloid beta-Peptides Base Sequence biology.organism_classification Molecular biology Peptide Fragments Recombinant Proteins Yeast chemistry Recombinant DNA Plasmids Biotechnology |
| Zdroj: | Protein Expression and Purification. 63:84-88 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2008.09.015 |
| Popis: | The human peptide rhA beta(1-42) was effectively produced through a novel expression system and purification procedure. The peptide rhA beta(1-42) was successfully expressed in Pichia pastoris, the methylotrophic yeast that has never been used as host. The cDNA encoding full-length hA beta(1-42) was synthesized with yeast bias codons and cloned into the pPICZ alpha A vector in frame with the yeast alpha-factor secretion signal under the transcriptional control of the AOX1 promoter and integrated into the secreting expression organism P. pastoris strain X33. Production of rhA beta(1-42) through fermentation was further optimized and scaled up in an 80 L fermentor. Secreted rhA beta(1-42) was purified using a two-step purification scheme: SP Sepharose ion exchange chromatography and source 30 RPC. The purification procedure is fast and efficient and reached a recovery of >93% without loss of activity. The purified rhA beta(1-42) was confirmed by Western blotting analysis and N-terminals amino sequencing analysis. This efficient and cost-effective expression system facilitates large-scale production and purification for recombinant rhA beta(1-42). |
| Databáze: | OpenAIRE |
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