Constructing R33A Mutant of HasAp Truncated Gene

The pET11a vector harboring a truncated gene of HasAp missing the last 21 C-terminal residues^{15 (link)} was used to construct the R33A mutant. The primers, synthesized by Integrated DNA Technologies, Inc. (Coralville, IA), were used with the QuickChange site-directed mutagenesis kit (Stratagene; La Jolla, CA). Primers used to introduce the R33A mutation were 5′-TATTTTGGCGATGTGAACCATGCGCCGGGCCAGGT-GGTGGATGGC-3′ and 5′-GCCATCCACCACCTGGCCCGGCGCATGGTTCACATCGCC-AAAATA-3′, where the underlined codons represent target substitutions. The recombinant gene harboring the mutation was transformed into XL1-Blue Competent cells (Stratagene) for amplification, and the DNA sequence verified by ACGT Inc. (Wheeling, IL). The recombinant DNA plasmid with the correct sequence was transformed into E. coli BL21-GOLD (DE3) competent cells (Agilent Technologies, CA) for subsequent protein expression. The protocols for protein expression, purification of the apo-proteins and reconstitution with heme, have been described previously.^{14 (link)}