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

这项研究的目的是开发利用重组DNA技术和亲和分离有效地生产多肽(小蛋白)的新方法。重点是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。这种多肽，人心钠素，将在大肠杆菌中与天冬酰胺酶蛋白融合表达。融合蛋白还将包含一个金属亲和结合位点(MABS)，它将用于选择性地从细胞碎片中去除融合蛋白。将评估两个不同的MAB和每个MAB在融合蛋白上的两个不同位置。融合蛋白的最终纯化将通过固定化D-天冬酰胺的亲和层析进行。这项拟议研究的意义在于，它可以导致以比目前更低的成本和更大的规模生产多肽的新工艺。这项研究还具有重要意义，因为它可以更好地理解如何在蛋白质上设计MABS，这对于蛋白质和多肽的纯化都是有用的。

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.