Pgex 6p 1 vector

For the construction of the pFLAG-SerRS, pFLAG-TRF1 and pcDNA6c-POT1 plasmids, human SerRS, TRF1 and POT1 genes were cloned from HEK293T cells by RT-PCR and inserted into the pFLAG-CMV2 vector (Sigma-Aldrich, St Louis, MO, USA) or the pCDNA6c vector (Thermo-Fisher Scientific, Waltham, MA, USA). For purifying recombinant SerRS and POT1, the coding sequences for SerRS and POT1 were subcloned into both the pET20b vector (Merck, Temecula, CA, USA) and the pGEX-6p-1 vector (GE Healthcare Bio-Sciences, Uppsala, Sweden).

Codon-optimized cDNA of 20 CorB orthologs were synthesized (Bio Basic Inc., Markham, Canada) and sub-cloned into NcoI and XhoI sites of pCGFP-BC vector^{57 (link)} with a C-terminal GFP-His8-tag for small-scale expression and detergent screening. Promising orthologs were subcloned into NdeI and XhoI sites of pET29a vector (Millipore Sigma) with a C-terminal His6-tag for large-scale expression. Constructs for M. thermophilus CorB (UniProt entry A0A1G8XA46): MtCorB (residues 1–426), MtCorBΔC (residues 1–322), MtCorBΔC_Δloop (residues 1–322 Δ259–262), and MtCorBΔC R235L. Construct for T. thermophilus CorB (UniProt entry A0A0K6IWT9): TtCorB (residues 1–445). For MtCorB constructs lacking TMD, MtCorB_CBS+CorC (residues 199–417), MtCorB_CBS (residues 199–322), and MtCorB_CorC (residues 333–417) were subcloned into BamHI and XhoI sites of pGEX-6P-1 vector (GE Healthcare) with an N-terminal GST-tag. QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) was used for the generation of point mutations. A list of mutagenic primers is included in Supplementary Table 1.

The DNAs encoding full-length CpCdc13A and CpCdc13B, as well as various domains (see Table 1 for the amino acids included in each expression construct), were amplified by PCR and cloned into the pSMT3 vector (21 (link)) or the pGEX6P-1 vector (GE Healthcare) to enable their expression as HIS₆-SUMO or GST fusion proteins, respectively. The pSMT3 vector was constructed by inserting the SUMO open reading frame in between the NheI and BamHI sites of pET-28a. Some reverse primers used for PCR contain the FLAG tag to enable purification and detection of the fusion protein through the FLAG antibody. Each HIS₆-SUMO fusion protein was expressed alone or co-expressed with a GST fusion protein in Escherichia coli BL21 (DE3). The growth and induction protocols as well as the extract preparation procedures were as previously described (12 (link)).

VWF73 mutants were cloned into the pGEX6P-1 vector (GE Healthcare Life Sciences, Piscataway, NJ) with a COOH-terminal 6X-His tag and an NH₃-terminal GST tag. Vectors were transformed into BL21 E. coli (Agilent, Santa Clara, CA), and peptide expression was induced for 2 hr with 1 mM IPTG upon reaching OD_600nm of 0.6 in a shaker flask grown at 30°C. Bacteria were lysed using Cell Lytic B (Sigma) in the presence of Complete Protease Inhibitors without EDTA (Roche). Peptide purification was performed using sequential nickel-NTA agarose and glutathione sepharose columns as previously described [15 (link)]. Four mL nickel NTA agarose was equilibrated with wash buffer (20 mM Tris pH 7.4, 600 mM NaCl, 40 mM imidazole, and 0.01% tween 20) before passing induced cell lysates over the column. The column was washed with 10 column volumes of buffer before eluting with TBS containing 250 mM imidazole. Samples were concentrated and buffer exchanged into TBS using 10 kDa cut-off filter Microcon centrifuge concentrators (EMD Millipore, Billerica, MA). Concentrated samples were then applied to a GST Spin-Trap column (GE Healthcare Life Sciences), washed with TBS, and eluted with 1 mM L-glutathione. Peptides were concentrated and buffered exchanged into TBS, and purity was analyzed by SDS-PAGE followed by staining with SYPRO RUBY (Invitrogen). Proteins were stored at -80°C until use.