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

这项研究的目的是开发新的 利用重组DNA技术和亲和性 分离以有效地产生肽（小蛋白）。 重点将放在改善两个复杂的部分， 纯化过程：1）去除细胞碎片，和2） 最后纯化至高纯度。 肽，人心房 利钠肽，将表达融合蛋白 天冬酰胺酶在细菌E.杆菌 融合蛋白 还含有金属亲和结合位点（MABS）， 将用于选择性地去除融合蛋白 从细胞碎片中分离出来 两种不同的MABS和两种不同的 将评估每个MAB在融合蛋白上的位置。 融合蛋白的最终纯化将通过亲和层析进行。 通过柱色谱法在固定化D-天冬酰胺上进行纯化。 这项研究的意义在于，它可以引导 涉及以显著更低的成本生产肽的新方法， 成本和规模比现在大得多。 研究 也很重要，因为它可以导致一个更好的 了解如何设计蛋白质的MABS， 可用于蛋白质以及肽的纯化。

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.