Research Initiation Award: Utilization of Genetic Engineering and Affinity Separations to Improve Peptide Production

本研究的目的是开发利用重组DNA技术和亲和分离有效生产肽（小蛋白）的新方法。重点将放在改进纯化过程的两个复杂部分：1)去除细胞碎片，2)最终纯化到高纯度。人心房利钠肽将在大肠杆菌中与蛋白天冬酰胺酶融合表达。融合蛋白还将含有金属亲和结合位点（MABS），该位点将用于选择性地从细胞碎片中去除融合蛋白。将评估两种不同的单抗和每个单抗在融合蛋白上的两个不同位置。融合蛋白的最终纯化将在固定化d -天冬酰胺上进行亲和层析。所提出的研究的意义在于，它可以导致以比目前低得多的成本和更大的规模生产肽的新工艺。这项研究的意义还在于，它可以更好地理解如何设计蛋白质上的单克隆抗体，这将有助于蛋白质和肽的纯化。

The objective of the proposed research is to develop new methods which utilize recombinant DNA technology and affinity separations to produce peptide (small proteins) effectively. Emphasis will be placed upon improving two complex parts of the purification process: 1) removal of cell debris, and 2) final purification to high purity. The peptide, human atrial natriuretic peptide, will be expressed fused to the protein asparaginase in the bacterium e. coli. The fusion protein will also contain a metal affinity binding site (MABS), which will be utilized in selectively removing the fusion protein from the cell debris. Two different MABS and two different locations of each MAB on the fusion protein will be evaluated. Final purification of the fusion protein will be by affinity chromatography on immobilized D-asparagine. The significance of the proposed research is that it can lead to new processes for producing peptide at significantly lower cost and on a much larger scale than at present. The research is also significant in that it can lead to a better understanding of how to design a MABS on proteins, which would be useful in the purification of proteins as well as peptide.