Improved Tools for Expression of Recombinant Genes in the Yeast Pichia pastoris

描述(申请人提供)：拟议研究的目标是鉴定和开发用于表达甲醇营养酵母巴斯德毕赤酵母的重组基因的新的基本工具。近年来，这种酵母已成为生产生物药物的重要宿主，已有5000多篇关于其用途的出版物，至少有两种人类生物药物(人血清白蛋白和ECallantide)在美国市场上上市，其他处于临床试验管道中的药物正处于不同阶段。该项目将由Biogrammatics公司部分或全部完成，该公司成立于2008年1月，是一家小型生物技术公司，其业务是开发、利用和销售在这种酵母中表达异源蛋白质的改进工具。Biogrammatics的商业战略包括两个总体目标：1)销售新的巴斯德酵母成分；2)为客户构建、测试和改进这种酵母的定制重组菌株。本申请有三个具体目标：1)在全基因组范围内寻找新的强启动子和/或调控良好的启动子，用于重组基因在巴斯德毕赤酵母中的表达；2)开发用于DNA介导性转化的巴斯德毕赤酵母合格细胞的改进方法，以及3)鉴定和开发改进的信号序列以指导从这种酵母中分泌重组蛋白如果该项目通过为期两年的第一阶段SBIR-Shift奖励机制提供资金，将产生生物语法学的产品，而不需要第二阶段。在该项目启动时，申请的首席PI，Cregg博士将从凯克研究生院的教授“转变”为生物语法学的员工。) 与公共健康相关：在9000亿美元的治疗剂市场中，蛋白质/多肽是增长最快的领域，有望实现真正的个性化药物和干细胞疗法。巴斯德毕赤酵母分泌启动子、转化效率和信号序列的优化将显著提高重组蛋白生产的成功率，从而加快rProtein治疗领域的临床试验和药物批准。

DESCRIPTION (provided by applicant): The goal of the proposed research is to identify and develop new basic tools for the expression of recombinant genes for the methylotrophic yeast Pichia pastoris. In recent years this yeast has become an important host for the production of biopharmaceuticals with over five thousand publications on its use and at least two human biopharmaceuticals (human serum albumin and ecallantide) on the market in the US and others at various stages in the clinical trial pipeline. This project will be done in part by and wholly for Biogrammatics, Inc. a small biotechnology company incorporated in January of 2008 whose business is to develop, utilize and sell improved tools for heterologous protein expression in this yeast. The business strategy of Biogrammatics involves two general objectives: 1) the sale of novel P. pastoris components and; 2) the construction, testing and improvement of custom recombinant strains of this yeast for customers. Three specific aims are outlined in this application: 1) to conduct a genome wide search for new strong and/or well regulated promoters for expression of recombinant gene in P. pastoris; 2) to develop improved methods for preparation of competent P. pastoris cells for DNA-mediate transformations and 3) to identify and develop of improved signal sequences to direct the secretion of recombinant proteins from this yeast The project, if funded through a two year Phase I SBIR-SHIFT award mechanism will result in products for Biogrammatics without the need for a Phase II. At the initiation of this project, the head PI on the application, Dr. Cregg will "shift" from being a professor at the Keck Graduate Institute to an employee of Biogrammatics. ) PUBLIC HEALTH RELEVANCE: Proteins/peptides are the fastest growing sector in 900 billion dollar therapeutic agent market and hold the promise for true personalized medicine and stem cell therapies. Optimization of Pichia pastoris secretory promoters, transformation efficiency and signal sequences will significantly increase the success rate for recombinant protein production, leading to faster clinical trials and drug approvals in the rProtein therapeutic arena.