Expression and Purification of Functionally Active Recombinant Human Alpha 1-Antitrypsin in Methylotrophic Yeast Pichia pastoris

Human alpha 1-antitrypsin (AAT) cDNA was obtained from HepG2 cell lines. After PCR and construction of expression vector pPICZα-AAT, human AAT was expressed in the yeast Pichia pastoris (P.pastoris) in a secretary manner and under the control of inducible alcohol oxidase 1 (AOX1) promoter. The amoun...

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Detalles Bibliográficos
Autores principales: Arjmand, Sareh, Bidram, Elham, Lotfi, Abbas Sahebghadam, Shamsara, Mehdi, Mowla, Seyed Javad
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Avicenna Research Institute 2011
Materias:
Original Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558188/
https://www.ncbi.nlm.nih.gov/pubmed/23408781
Descripción
Sumario:Human alpha 1-antitrypsin (AAT) cDNA was obtained from HepG2 cell lines. After PCR and construction of expression vector pPICZα-AAT, human AAT was expressed in the yeast Pichia pastoris (P.pastoris) in a secretary manner and under the control of inducible alcohol oxidase 1 (AOX1) promoter. The amount of AAT protein in medium was measured as 60 mg/l 72 hr after induction with methanol. Results indicated the presence of protease inhibitory function of the protein against elastase. Purification was done using His-tag affinity chromatography. Due to the different patterns of glycosylation in yeast and human, the recombinant AAT showed different SDS-PAGE patterns compared to that of serum-derived AAT while pI shifted from 4.9 in native AAT compared to 5.2 in recombinant AAT constructed in this study.

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