期刊
BIOTECHNOLOGY PROGRESS
卷 22, 期 5, 页码 1416-1425出版社
AMER CHEMICAL SOC
DOI: 10.1021/bp060111n
关键词
-
This paper presents a microchip-based system for collecting kinetic time-based information on protein refolding and unfolding. Dynamic protein conformational change pathways were studied in microchannel flow using a microfluidic device. We present a protein-conserving approach for quantifying refolding by dynamically varying the concentration of the chemical denaturants, guanidine hydrochloride and urea. Short diffusion distances in the microchannel result in rapid equilibrium between protein and titrating solutions. Dilutions on the chip were tightly regulated using pressure controls rather than syringe-based flow, as verified with extensive on-chip tracer dye controls. To validate this protein assay method, folding transition experiments were performed using two well-characterized proteins, human serum albumin (HSA) and bovine carbonic anhydrase (BCA). Transition events were monitored through fluorescence intensity shifts of the protein dye 8-anilino-1-naphthalenesulfonic acid (ANS) during dilutions of protein from urea or guanidine hydrochloride solutions. The enzymatic activity of refolded BCA was measured by UV absorption through the conversion of p-nitrophenyl acetate (p-NPA). The microchip protein refolding transitions using ANS were well-correlated with conventional plate-based experiments. The microfluidic platform enables refolding studies to identify rapidly the optimal folding strategy for a protein using small quantities of material.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据