Recombinant Expression of Human DNPH1 Proteins

The DNA-encoding HsDNPH1
(UniProt O43598) and a truncated HsDNPH1 (HsDNPH1^Trunc), each containing a TEVP-cleavable N-terminal His-tag
with restriction sites for NdeI and HindIII at the 5′- and
3′-ends, respectively, were purchased as codon-optimized (for
expression in E. coli) gBlocks (IDT). HsDNPH1- and HsDNPH1^Trunc-encoding
gBlocks were polymerase chain reaction-modified and inserted into
modified pJexpress414 plasmids using Gibson Assembly^{12 (link)} according to the manufacturer’s instructions (New
England Biolabs). Constructs were used to transform E. coli DH5α-competent cells (New England Biolabs),
were subsequently sequenced (Eurofins) to confirm the insertion of
the gene and that no mutations had been introduced, and then used
to transform E. coli BL21(DE3)-competent
cells (New England Biolabs). Transformed cells were grown independently
in lysogeny broth containing 100 μg mL^–1 ampicillin
at 37 °C until an optical density at 600 nm (OD₆₀₀) of 0.6–0.8 before expression was induced with 0.5 mM IPTG.
Cells were grown for an additional 3 h, harvested by centrifugation
(6774g, 15 min, 4 °C), and stored at −20
°C.