Plasmid construction, protein recombination, and purification

Mammalian expression vectors for full-length LINK-A and the deletion mutant were constructed by subcloning the gene sequences into a pCDNA3.1 (+) backbone and pInducer20 inducible lentiviral expression vector (Life Technologies). Mammalian expression of full-length TRIM71, PKA C-α, CNR2, GABR1, ADA2A, ACM4, OPRM, and mutant vectors were constructed by subcloning the corresponding gene sequences into the His-tagged expression vector (pcDNA™-DEST40) using the Gateway system (Life Technologies). Bacteria expression of full-length CNR2, GABR1, ADA2A, ACM4, OPRM, and mutant vectors was constructed by subcloning the corresponding gene sequences into the GST-tagged expression vector pGEX-5X-1. All single-point and deletion mutations were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Recombinant proteins were expressed in the Escherichia coli strain BL21-CodonPlus (DE3)-RIPL (Agilent Technologies) and purified using the Protein Purification Kit (Clontech).