African Journal of Biotechnology Vol. 5 (4), pp. 321-326, 16 February 2006   

ISSN 1684–5315 © 2006 Academic Journals        

Full Length Research Paper

Subcloning and expression of human alpha-fetoprotein gene in Pichia pastoris

Mohammad Reza Mashayekhi^1,3, Nosratollah Zarghami^1,3*, Delavar shahbazzadeh², farzin Rohvand², Mohammad Azizi² and Behrang Alani^1,3

¹Department of Molecular Biochemistry and RIA, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

²Department of Biotechnology, Pasteur Institute, Tehran, Iran.

³Tuberclosis and Lung Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran.

*Corresponding author. E-mail: nzarghami@hotmail.com.

Accepted 11 November, 2005

Alpha-fetoprotein protein (AFP) is naturally found in fetal serum while production and appearance after birth is indicative of presence of malignant tumors. Therefore, by measuring this protein during fetal period and after birth, it is possible to diagnose abnormalities and tumors in fetus or the newborn. The objective of this study was to produce and purify AFP protein using DNA recombinant technology to apply in diagnostic kit preparations. In this study Pichia pastoris as metylotrophic yeast was used for AFP production. After construction of recombinant plasmid, pS1-AFP, electroporation and lithium chloride techniques were used for transferring to susceptible cells. The quantity and quality of the produced protein were checked by SDS-PAGE and ELISA methods. Selection of transformed mutant strains of Mut^s and culturing them in glycerol media (YPG) up to OD₆₀₀=6 and their transfer to methanol media (YPM) with augmentation of methanol to 1% final concentration resulted in inducing protein production in auxotrophic media lacking histidine. This protein could be useful in monoclonal antibody production and in diagnostic kit preparations.

Key words:  Alpha-fetoprotein, Pichia pastoris, cloning, expression.