The genes HiGulD (Uniprot ID Q57517) and HiUxuA (Uniprot ID P44488) were amplified from H. influenzae strain Rd KW20 (ATCC 51907) genomic DNA. PCR was performed using KOD Extreme DNA Polymerase (Novagen) according to the manufacturer’s guidelines. The conditions were: 2 min at 95°C, followed by 40 cycles of 20 s at 95°C, 20 s at 66°C, and 20 s at 72°C. Primers are listed in Supplementary file 7 . The amplified fragment was cloned into the C-terminal TEV cleavable 10x-Histag containing vector pNYCOMPS-LIC-TH10-ccdB (C-term) such that the tag is out of frame (pNYCOMPSC-tagless), by ligation-independent cloning (Aslanidis and de Jong, 1990 (link); Savitsky et al., 2010 (link)).
The pNYCOMPSC-tagless HiGulD and HiUxuA constructs were transformed into E. coli BL21 (DE3) for expression. Both HiGulD and HiUxuA were purified from 1 L of culture using DEAE Sepharose, Q‐Sepharose, and phenyl-Sepharose columns (all Amersham Biosciences) as previously described (Wichelecki et al., 2014 (link)). Proteins were concentrated to 15 g/L and 6 g/L, respectively, flash frozen in liquid nitrogen, and stored at −80°C.
The pNYCOMPSC-tagless HiGulD and HiUxuA constructs were transformed into E. coli BL21 (DE3) for expression. Both HiGulD and HiUxuA were purified from 1 L of culture using DEAE Sepharose, Q‐Sepharose, and phenyl-Sepharose columns (all Amersham Biosciences) as previously described (Wichelecki et al., 2014 (link)). Proteins were concentrated to 15 g/L and 6 g/L, respectively, flash frozen in liquid nitrogen, and stored at −80°C.
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