Rapid screening of elution conditions prior to immunopurification of proteins.

Attempts are frequently made to purify the desired proteins to the state of homogeneity by using an immunoaffinity column chromatography. The readymade immunoaffinity supports that are used in the process offer rapid and high-efficiency coupling of ligands, and are commercially available (1). Most of the laboratory work is spent testing the conditions that will ensure a highly efficient elution of active proteins. Since the availability of a ligand or an antibody is often limited, it is important to develop a procedure that will consume minimum amounts of those reagents and allow for a multiple use of the immunoaffinity column. A typical screening procedure involves a series of pilot purifications, which use different types of increasingly strong elution agents. We are proposing an alternative, a simple, fast method to determine the efficiency of elution of an antigen from the immunoaffinity column before the actual purification run. To elute active proteins with high recovery, it is essential to compromise between the harsh or even denaturing conditions that usually yield inactive products and the mild ones that preserve both the antibody and the ligand in an active state, but are characterized as low-efficient. We have tested several elution conditions, listed below, and a more complete list of suggested elution reagents can be found elsewhere (1). The enzyme selected for the exemplary immunopurification described here was restriction endonuclease CviJI, derived from an IL-3A phycodnavirus-infected Chlorella and known to be sensitive to inactivation (3). We used the following steps in the immunoscreening procedure. Step 1. The monoclonal antibody column was prepared with the use of activated resin Affi-Gel 10 Immunoaffinity Support (Bio-Rad, Hercules, CA, USA). The support was mixed with the 3CD2 anti-CviJI monoclonal antibodies and processed as instructed by the manufacturer. Step 2. Eluting solutions, which might successfully work, were prepared: (a) TBST [50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 5 mM sodium azide, 0.1% Tween 20 (20 mL)]; (b) 5 M LiCl in 10 mM HEPES, pH 7.0; (c) 1 M guanidine HCl; (d) 2 M guanidine HCl; (e) 1 M urea; (f) 2 M urea, (g) 2 M MgCl2, 20 mM HEPES, pH 6.0; (h) 3.5 M MgCl2, 20 mM HEPES, pH 6.0; (i) 100 mM glycine, pH 2.5; (j) 50% ethylene glycol, 10% glycerol, 3 mM TrisHCl, pH 8.0; and (k) deionized water. Step 3. Rectangular pieces of nitrocellulose membrane (Whatman International, Maidstone, Kent, England, UK) were cut, approximately 5.5 cm long and 0.5 cm wide (0.25 cm2 per one eluting agent). One extra square was added as a control of detection reaction. At this stage, it was possible to choose the composition of incubation buffer and temperature, which would preserve the native state of antigens. The pieces of membrane were incubated for 30 min at 4°C with the CviJI (3) antigen solution [recombinant CviJI endonuclease at a concentration of 2.3 μg/mL in 10 mL of buffer: 20 mM Tris-acetate, pH 7.2, at 25°C, 0.5 mM EDTA, 0.1 mM dithiothreitol (DTT), 50 mM K OAC, 5 mM Mg OAC, 10% glycerol]. In general, once an antigen is immobilized, its thermal stability increases. Thus, membranes were treated at room temperature (RT). The benefit of higher Benchmarks