Ptrchis topo ta expression kit

All constructs used in this study were confirmed by DNA sequencing at the Oklahoma Medical Research Foundation DNA Sequencing Facility. The CnYPD1 gene was PCR amplified from cDNA (kindly provided by Dr Jan Fassler, University of Iowa) into a pTrcHis-TOPO vector using the pTrcHis TOPO^® TA Expression Kit (Life Technologies, Carlsbad, California, USA) to construct the plasmid trcHis-CnYPD1. The pTrcHis-CnYPD1 plasmid served as a template for making a series of N-terminal truncation constructs (Δ5, Δ19, Δ43, Δ50, Δ60, Δ77, Δ83 and Δ100). The hypothetical phospho-accepting histidine residue of CnYpd1, H138, was mutated to a glutamine residue by site-directed mutagenesis PCR using the pTrc-CnYPD1 plasmid as a template. Three mutants were constructed using TrcHis-CnYPD1 as a template in order to determine which specific residues are involved in metal ion binding, E58A, E49A-E54A and D60A-E67A. CnYpd1-E58A was constructed using site-directed mutagenesis PCR. The mutants E49A-E54A and D60A-E67A were constructed using ligation-independent cloning (Scholz et al.2013 (link)).

All chemicals for protein expression and purification were of ultrapure grade from Ameresco^® (Framingham, Massachusetts, USA) or Sigma-Aldrich^® (St. Louis, Missouri, USA). The pTrcHis-TOPO^® TA Expression Kit was purchased from Life Technologies (Carlsbad, California, USA) for gene cloning. Ni-NTA affinity resin was purchased from McLab (San Francisco, California, USA). HiTrapQ columns were purchased from GE Healthcare Life Sciences (Pittsburgh, PA, USA) and were used on an AKTÄ prime chromatography system from GE Healthcare. [γ-³²P] ATP (3000 Ci/mmol) was purchased from Perkin Elmer (Waltham, Massachusetts, USA). TEV protease was produced in our laboratory from an expression plasmid that was kindly given to us by J. Doudna at University of California, Berkeley (Lucast, Batey and Doudna 2001 (link)).