Dept. of Science & Engineering
Oregon Health & Science University
This thesis describes the development of a more efficient scheme for multiple copy selection and a new selectable marker in yeast, both of which add to the arsenal of tools used for gene expression in Pichia pastoris. Transformation and integration of the expression vector, and then gene expression to produce a high protein yield is a key objective in using P. pastoris as a host expression system. Because of its growing popularity for such use, the development of new markers and methods are essential to support this trend. This thesis describes a novel scheme for the enrichment of multiple copy clones. We demonstrate that a transformant with a single or low copy number of the expression vector can be induced to amplify its vector copy number. The resulting clones selected after this posttransformational vector amplification (PTVA) process have been shown to consist of a tandem arrangement of full vector copies integrated into the genome. The PTVA process proved to be more convenient and efficient than previous methods. The development of a new marker system for the transformation of P. pastoris utilizes the formaldehyde dehydrogenase gene (FLD1) as a selectable marker is also described in this thesis. We show that a P. pastoris fld1 mutant strain can be transformed with normal efficiency using the wild type FLD1 gene as a selectable marker. There are two benefits of this marker system which provide researchers with an excellent alternative for the expression of their gene. The first advantage is that FLD1 is endogenous to P. pastoris. This allows for the construction of an expression vector that contains no exogenous sequences, except for the foreign gene insert, upon transformation in the yeast. The lack of bacterial sequences upon transformation is important in the regulation of proteins expressed for therapeutic purposes. The second valuable feature of this marker system is the ability to enhance multiple copies in transformed strains. Studies have shown that increasing copy number of the expression vector is often directly proportional to the increase in protein production. These characteristics can prove to be a significant advantage for researchers in academic and industrial laboratories looking for an alternative marker system.
Div. of Environmental & Biomolecular Systems
School of Medicine
Sunga, Anthony Jay J., "Novel Methods for Generating Strains with Increased Copy Number in the Yeast Pichia pastoris" (2008). Scholar Archive. 348.