http://myais.fsktm.um.edu.my/2283/ Integration of the Toxoplasma gondii MIC2 Gene Into the Pichia pastoris Genome Yee, Wai-Yan, Wah, Kiew-Lian, Q Science, Computer Science R Medicine, Dentistry, Pharmacy, Nursing The Toxoplasma gondii MIC2 gene encodes a microneme protein that is believed to play an important role in the adhesion of the parasite to its host cell. Since the Pichia pastoris expression system is capable of producing biologically active foreign proteins, expression of recombinant MIC2 in this yeast will potentially provide useful MIC2 material for functional studies of this protein. As a prior effort to utilise this eukaryotic expression system in the study of MIC2, we have integrated the MIC2 gene into the genome of P. pastoris. Amplification of the MIC2 gene was carried out using the polymerase chain reaction (PCR) technique and cloned into the pCR2.1 vector. The resulting pCR2.1-MIC2 recombinant plasmid was transformed into the Escherichia coli TOP 10 strain. Positive colonies were determined by DNA plasmid analysis. The pCR2.1-MIC2 recombinant plasmid was isolated, and digested with EcoRI and XbaI restriction enzymes. The MIC2 fragment was then purified and cloned into the pPICZ B expression vector. The resulting pPICZ-MIC2 recombinant plasmid was transformed into the E. coli DH5α strain. Positive transformants were determined by PCR analysis. The pPICZ-MIC2 recombinant plasmid was extracted, linearised and transformed into P. pastoris via electroporation. The integration of MIC2 into the P. pastoris genome was subsequently demonstrated by PCR. Malaysian Society for Biochemistry and Molecular Biology 2005 Journal PeerReviewed http://ejum.fsktm.um.edu.my/ArticleInformation.aspx?ArticleID=622 Yee, Wai-Yan, and Wah, Kiew-Lian, (2005) Integration of the Toxoplasma gondii MIC2 Gene Into the Pichia pastoris Genome. Malaysian Journal of Biochemistry and Molecular Biology, 11 (1). pp. 42-45. ISSN ISSN 1511-2616 http://myais.fsktm.um.edu.my/2283/