SlideShare for iOS
by Linkedin Corporation
FREE - On the App Store
Recombinant DNA technology is major DNA-based tool that has gained popular attention in the past decade. Significant advances in the development of new strains and vectors, improved techniques, and ...
Recombinant DNA technology is major DNA-based tool that has gained popular attention in the past decade. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of yeast species have led the recombinant yeast technology a robust tool for both designed catalysts and new biologicals. Yeast combines molecular genetic manipulations and growth characteristics of prokaryotic organisms together with the sub-cellular machinery for performing post-translational protein modifications (O and N- linked glycosylation, disulphide bond formation) and secretion of protein (Intracellularly or extracellularly). A large number of yeast hosts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, etc) are available for heterologous protein expression. The methylotrophic yeast, Pichia species is the most highly developed one among a small group of alternative yeast species chosen for their perceived advantages over S. cerevisiae as a expression host for the generation of recombinant protein of commercial interest. Advantages of the system include the AOX1 promoter (alcohol oxidase) and other alternate promoters (GAP, FLD1, PEX8, and YPT1), with transcription characteristics that are useful for regulating heterologous protein expression.
Auxotrophic mutants (MutS and Mut+) and a new set of biosynthetic markers such as ADE1, ARG4 and URA 3 have been used successfully for better selection of transformed host. Protease deficient hosts and site specific integration of expression vectors into Pichia genome result into high expression of gene of interest. Additional features that are present in certain P. pastoris expression vectors serve as tools for specialized functions. The availability of the expression system as a commercially available kit (Invitrogen) extends the usefulness of system. Several different secretion signal sequences including the native secretion signal or secretion signal sequences from S. cerevisiae such as µ factor prepro peptide causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification. The P. pastoris expression platform is now well developed, as proven by multiple products used in human and veterinary medicine and in industry. A better understanding of secretion signals, glycosylation, and endogenous yeast proteases would be extremely helpful in developing and improving the yeast heterologous expression system.