Genetic Engineering of Proteins to Enhance Product Recovery

利用分子生物学赋予一种产品的特性，使其能够从多组分细胞提取物中选择性去除，这对生物技术产品的下游加工，包括酶、激素和抗体，具有巨大的好处。理论和过去的结果都表明，在蛋白质上添加带电荷的尾部有助于分离方法的选择性。该方案旨在将多肽尾部与蛋白质融合，以增强蛋白质的恢复。β -半乳糖苷酶被选为模型蛋白来实现这一建议。工作计划包括以下内容：多肽尾部的设计。2. 尾巴基因片段的合成。3. 将该片段克隆为-半乳糖苷酶基因。4. 基因在大肠杆菌中的表达。5. 评估靶蛋白的结构和/或活性变化。6. 沉淀法和两相水萃取法回收融合蛋白。这项拨款申请是我们希望在工程理事会的生物技术计划中看到的跨学科提案类型的一个很好的例子。首席研究人员被认为完全有资格进行拟议的研究。

Using molecular biology to bestow a characteristic on a product that permits its selective removal from a multicomponent cellular extract can be of immense benefit in the downstream processing of the products of biotechnology, including enzymes, hormones and antibodies. Theory and past results both indicate that the addition of charged tails to proteins can aid the selectivity of separation methods. This proposal seeks to fuse polypeptide tails to proteins in order to enhance the recovery of proteins. Beta-galactosidase has been chosen as a model protein to carry out this proposal. The work plan includes the following: 1. Design of the polypeptide tail. 2. Synthesis of the gene segment for the tail. 3. Cloning this segment into the beta-galactosidase gene. 4. Expression of the gene in E. coli. 5. Assessment of structural and/or activity changes in the target protein. 6. Recovery of the fused protein by precipitation and two-phase aqueous extraction. This grant application is an excellent example of the type of cross-disciplinary proposal we wish to see in the Engineering Directorate's Biotechnology Program. The Principal Investigators are considered to be well qualified to carry out the proposed research.