AOX1 PROMOTER · Purification and Characterization of Regulatory Proteins of the Alcohol Oxidase 1 (AOX1) Promoter of Pichia pastoris

Purification and Characterization of Regulatory Proteins of the Alcohol Oxidase 1 (AOX1) Promoter of Pichia pastoris

P. pastoris has been used extensively for recombinant protein expression for a variety of applications, ranging from food enzymes to pharmaceuticals. It is also used as a model organism to study eukaryotic gene expression and peroxisome biogenesis. The regulation of the AOX1 promoter of P. pastoris has gained much attention due to widespread use of the promoter for recombinant protein expression. Although the AOX1 promoter is one of the most commonly used promoters for recombinant expression in the Pichia expression system, the regulation of its induction and repression mechanism has not been fully elucidated. To date, only the methanol expression regulatory protein 1 (Mxr1p) along with its regulator, a 14-3-3 family protein, have been identified as activators of the methanol utilization pathway in P. pastoris. The primary objective of this project is to identify the regulatory sequences on the AOX1 promoter and to purify and characterize the regulatory proteins. Under this primary objective, specific objectives of the project are to purify the regulatory proteins and generate a new host strain which both over-expresses the activator protein and suppresses the expression of repressor proteins. We have purified a protein complex that binds to C fragment of the AOX1 promoter in vitro. Biotin-labeled C fragment was immobilized to streptavidin magnetic beads which were used as affinity column for purification of a complex protein. The purified proteins contained two subunit of pyruvate dehydrogenase complex (PDH). In yeast PDH complex consists of five subunits, namely, E1-alpha, E1-beta, E2, and E3 and E3BP. We have attempted to make deletion mutants of P. pastoris for each PDH subunit. We were able to generate E3BP mutant strain, however, attempts to delete the other subunits of PDH complex failed. E1-beta subunit which is responsible for the PDH catalitic activity and E3BP were over-expressed and purified by his-tag affinity purification. The purified proteins did not form a complex with the AOX1 promoter by Electrophoretic Mobility Shift Assay (EMSA). We have identified an essential protein in the methanol utilization pathway in P. pastoris. The newly identified protein was named Methanol eXpression Regulatory protein 2 (Mxr2p). Deletion of the MXR2 gene resulted in loss of growth in minimal methanol media. However, the growth on glucose and glycerol was not affected. Mxr2p is a Zn(II)2Cys6-type zinc finger protein and shares homology to H. polymorpha (P. angusta) Mut3p and C. boidini Trm1p. The MXR2 transcript was detected only in cells growing on methanol and was localized in the nucleus as shown by florescence microscopy. We have also isolated two repressor proteins (Mig1 and Mig2) which have effect on the Aox activity in P. pastoris grown in glucose and glycerol containing media. We have tested deletion of the repressor proteins and over-expression of the activator protein with Enhanced green fluorescent protein (EGFP) and Aspergillus niger Xylanase (XylB). Secretion of the EGFP and Xylanase were increased about two fold in strain over-expresses the Mxr2p under the GAP promoter and lacking Mig1p and Mig2p. This strain can be used to expresses recombinant proteins at large scales.

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