Recombinant Human 1-acyl-sn-glycerol-3-phosphate acyltransferase delta (AGPAT4)

To produce a recombinant human 1-acyl-sn-glycerol-3-phosphate acyltransferase delta (AGPAT4) protein tagged with an N-terminal 10xHis-tag in an in vitro E. coli expression system, the full-length human AGPAT4 gene fragment (1-319aa) along with an N-terminal 10xHis-tag is cloned into a suitable expression vector with the necessary regulatory elements. The recombinant plasmid DNA containing the AGPAT4 gene fragment is isolated and purified. In vitro transcription is performed using the purified plasmid DNA to synthesize mRNA, which serves as a template in the subsequent in vitro translation reaction. The translation reaction is set up using an E. coli cell-free extract supplemented with energy sources and amino acids. Following incubation, the recombinant AGPAT4 protein is purified from the cell culture medium. The purity of the recombinant AGPAT4 protein, measuring up to 85%, is assessed by SDS-PAGE analysis, where it appears as a band with a molecular weight of approximately 40 kDa.