[Prokaryotic expression of human Alg1 protein and analysis of the transmembrane domain properties].

As the most common type of protein glycosylation, <i>N</i>-glycosylation begins with the synthesis of the dolichol-linked oligosaccharide (DLO) precursor in the endoplasmic reticulum. The mannosyltransferase Alg1 catalyzes the addition of the first mannose molecule to DLO, serving as a key enzyme in this biochemical pathway. The defect of human <i>ALG1</i> gene can lead to the congenital disorders of glycosylation (CDG), i.e., ALG1-CDG. Therefore, it is of great significance to establish the expression and activity assay system of <i>Homo sapiens</i> Alg1 (<i>Hs</i>Alg1) <i>in vitro</i>. In this study, full-length plasmid pET28a-His6-<i>Hs</i>Alg1 and transmembrane domain-lacking plasmid pET28a-His6-<i>Hs</i>Alg1_23-464 were constructed and expressed in <i>Escherichia coli</i>, and the activity of recombinant <i>Hs</i>Alg1 and <i>Hs</i>Alg1_23-464 was measured by liquid chromatography tandem mass spectrometry (LC-MS) with dolichyl-pyrophosphate GlcNAc2 (DPGn2) as the substrate. The results showed that <i>Hs</i>Alg1 had transglycosylation activity, while the activity decreased after protein purification, which was partially restored upon re-addition of membrane components. However, <i>Hs</i>Alg1_23-464 was unable to catalyze glycosylation. The results indicate that the N-terminal transmembrane domain (TMD) of <i>Hs</i>Alg1 plays an important role in the catalytic reaction. This study lays a foundation for further expression and activity analysis of ALG1-CDG-related mutants.