Increasing Affinity of Interferon- Receptor
1 to Interferon- by Computer-Aided Design
Figure 3
Purification of monomeric refolded recombinant 6xHis-tagged IFN--Rx protein. (a) Typical chromatogram from separation of affinity-purified and refolded IFN--Rx variants by gel permeation chromatography on Superdex 200 10/300 GL as described in Section 2. Fraction 6, containing the monomeric forms of refolded IFN--Rx, was used for SPR measurements. (b) Analysis of purified soluble IFN--Rx on 12.5% SDS-PAGE under nonreducing conditions. Proteins were extracted in 8 M urea from inclusion bodies and purified by metal affinity chromatography on Ni-NTA agarose as described in Section 2. Upon refolding by dialysis against urea-free buffer the monomeric fraction was separated as outlined above. IFN--Rx with C-terminal His-Tag migrates at a molecular mass of 23 kDa when analyzed on non-reducing and at 27 kDa on reducing SDS-PAGE (not shown). Protein constructs are numbered as in Table 2.
