Prokaryotic expression of Hmbs missense mutations

A ~1.4 kb wild-type murine Hmbs cDNA (RefSeq {"type":"entrez-nucleotide","attrs":{"text":"NM_013551.2","term_id":"159110430","term_text":"NM_013551.2"}}NM_013551.2) was subcloned into the pET16b vector (Novagen, Madison, WI, USA) and designated pET-WT. Mutant constructs carrying the c.500G>A (p.Arg167Glu), c.499C>T (p.Arg167Try) or c.518_519GC>AG (p.Arg173Glu) substitutions, designated pET-R167Q, pET-R167W and pET-R173Q, respectively, were generated using the Qiagen Quickchange site-directed mutagenesis kit (Valencia, CA, USA) and primer pairs provided in the Supplementary Material, Table S5. The constructs were confirmed by sequencing and transformed into E. coli BL21 cells (Millipore Sigma, St. Louis, MO, USA). Following growth in LB medium, induction with 1mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and inoculation for 4 h, the cells were lysed and the supernatants were used to assay HMBS activities as previously described (39).