Protein Crystallographic Construct Design

The Protein Crystallographic Construct Design (ProteinCCD) metaserver was used to choose fragments from different domains of yeast and lancelet healing enzymes based on information acquired from various prediction servers for secondary structure, disorder, coiled coils, transmembrane segments, conserved domains, and domain linkers [34 (link)]. Multiple expression constructs encoding the PNK and CPDase domains of yeast tRNA ligase and full-length lancelet PNK/CPDase were assembled by sequence and ligation independent cloning (SLIC). The primers used are listed in (Additional file 1). Briefly, the DNA fragments of interest were amplified by polymerase chain reaction (PCR) from pET28a and pET20b plasmids harbouring ScTrl1 and BfPNK/CPDase, respectively [8 (link)]. The amplified products and linearized pET-his3C-LIC-amp vector (a kind gift from the Netherlands Cancer Institute) were separately treated with T4 DNA polymerase. The insert and vector were annealed, and the resulting plasmid was used to transform E. coli NEB5α cells (New England Biolabs, Germany). Transformed colonies were screened by colony PCR for recombinant plasmids that were purified, verified by sequencing, and used for protein expression.