Bailey S M, Blum P H, Meagher M M
Department of Biological Systems Engineering, School of Biological Sciences, University of Nebraska, Lincoln 68583-0919, USA.
Biotechnol Prog. 1995 Sep-Oct;11(5):533-9. doi: 10.1021/bp00035a006.
Pretreatment of recombinant Escherichia coli, expressing human growth hormone inclusion bodies, with guanidine hydrochloride and Triton X-100 prior to high-pressure homogenization has been investigated. Homogenates were analyzed for protein release, viscosity, and particle size. We were able to reduce the number of passes required for cell disruption and the number of downstream processing steps required for the recovery of protein from inclusion bodies by pretreating cells with guanidine HCl and Triton X-100. Pretreatment of exponential growth phase cells with 1.5 M guanidine HCl and 1.5% Triton X-100 gave adequate disruption after one pass at 41 MPa with a particle size distribution similar to that for untreated cells disrupted after one pass at 62 MPa. This combination of guanidine HCl and Triton X-100 was also selected so as to wash the inclusion bodies without solubilization of the human growth hormone. Pretreatment of cells with 4 M guanidine HCl produced cell debris that was substantially smaller than the debris from untreated cells and partially solubilized the inclusion bodies. Cells harvested in the stationary growth phase were more resistant to high-pressure homogenization and pretreatment.
研究了在高压匀浆之前，用盐酸胍和 Triton X - 100 对表达人生长激素包涵体的重组大肠杆菌进行预处理的情况。对匀浆产物进行了蛋白质释放、粘度和粒径分析。通过用盐酸胍和 Triton X - 100 预处理细胞，我们能够减少细胞破碎所需的通过次数以及从包涵体中回收蛋白质所需的下游加工步骤数量。用 1.5 M 盐酸胍和 1.5% Triton X - 100 对指数生长期细胞进行预处理后，在 41 MPa 下通过一次即可实现充分破碎，其粒径分布与未处理细胞在 62 MPa 下通过一次破碎后的粒径分布相似。选择盐酸胍和 Triton X - 100 的这种组合还为了在不溶解人生长激素的情况下洗涤包涵体。用 4 M 盐酸胍预处理细胞产生的细胞碎片比未处理细胞产生的碎片小得多，并且使包涵体部分溶解。在稳定生长期收获的细胞对高压匀浆和预处理更具抗性。
Zotero 插件