Figure 3.

(A) Expression constructs for human b5+b5R protein and its derivatives. The constructs for b5+b5R fusion as well as b5* and b5R* single-domain proteins were made by inserting PCR fragments into the pET-19b vector (Novagen), using NdeI site at the NH₂ end and BamHI site at the COOH end (both sites were included in PCR primers). The b5* domain extends from the NH₂ terminus through the NH₂-PSYPSF-COOH peptide sequences, and the b5R* domain from the NH₂-GQHVYLK-COOH peptide sequences through the COOH terminus of b5+b5R. The b5+b5R.ΔH was constructed by ligating the following DNA pieces: NdeI–NheI fragment for b5*, NheI–BamHI fragment for b5R*, and NdeI–BamHI fragment for pET19b vector. The presence of His₁₀ tag and enterokinase cleavage site at the NH₂ terminus of recombinant protein is indicated. (B) Light absorption spectrum for the human b5+b5R protein. Spectrum was measured at room temperature under air in low-salt buffer, either with 2 μM b5+b5R alone (oxidized) or mixed with additional 0.1 mM NADH (NADH-reduced). Excess NAD(P)H was necessary to maintain the b5+b5R protein in reduced state under aerobic conditions.