Rapid Purification and Characterization of Recombinant Proteins and Antibodies: Capturem High-Capacity Membranes

摘要

Recombinant protein production is immensely important in many research settings, including academic research institutions, biopharmaceutical organizations, and enzyme and agricultural industries. Fusion tags are widely used to improve yields and enable purification and characterization of protein structure and function. Polyhistidine tags, which incorporate 6-10 histidines at either terminus of the target proteins, are the most popular tag used for purification. The affinity of the histidines to immobilized metal ions such as Co2+ and Ni2+ is utilized to selectively bind the tagged construct to the matrix, while washing away unwanted materials, before eluting the target with low pH or imidazole-containing buffers. Typical purification methods using immobilized metal affinity chromatography (IMAC) columns take several hours to complete due to long column equilibration/binding times and slow diffusion of large macromolecules through the resin bed. The long times incre ase the risk of proteolytic degradation and activity loss due to unfolding or denaturation. Membrane-based affinity systems have rapid flow-induced mass transport and short residence times; however, they have been plagued with low capacity due to small internal surface areas. Here, we describe a novel, nylon-membrane-based IMAC system with chemically enhanced surface area of the pores that allow protein binding capacities comparable to, or better than, resins at 75 mg or more per cm3 of membrane. Unlike traditional resin-based systems, the entire purification process-from loading the lysate to eluting pure protein-can be completed at room temperature in less than five minutes. We have assembled these membranes into spin columns and filtration devices and demonstrate that their ability to purify his-tagged proteins produced in bacterial and mammalian cells. The millisecond residence time of the proteins on the membrane during binding minimizes the possibility of degradation. These membranes function perfectly in the presence of additives such as ethylenediaminetetraacetic acid (EDTA), reducing agents such as dithiothreitol (DTT), and under denaturing conditions (in the presence of urea and guanidium hydrochloride). We have extended the high-capacity membrane technology to immobilize Protein A and G, enabling extremely fast purification of antibodies from various matrices based on the affinity of these proteins for the fragment crystallizable region (Fc) region of antibodies. Antibody purification can be accomplished in less than 10 minutes, with capacities of up to 75 mg/ml or more, far exceeding the capacity of resin-based columns.