title: A simple and rapid strategy for the molecular cloning and monitoring of mouse HtrA2 serine protease.
abstract: A simple and rapid strategy for molecular cloning using a gel-free and antibiotic selection method is described which allows for the complete elimination of DNA extraction by gel electrophoresis, and thus has several advantages over gel-based cloning methods, including: (i) a cloning efficiency that is approximately 10-times higher due to the prevention of ethidium bromide ultraviolet-induced DNA damage and contamination with ligase inhibitors; (ii) the amount of plasmid DNA required is approximately five times less; and (iii) the cloning time is several hours less. Once the target gene, such as mouse HtrA2 serine protease, was cloned into the pEGFP-N3 plasmid, the integrity of the kanamycin-resistant molecular clone encoding the GFP fusion protein was verified by immunoblot and immunofluorescence assays. In addition, the integrity of the ampicillin-resistant molecular clone was directly evaluated by analyzing the expression and affinity purification of the GST fusion protein and by measuring its enzymatic activity. Therefore, this method is suitable for the routine construction of a plasmid expressing the gene of interest, and the usefulness of this strategy can be demonstrated by monitoring the expression of the target gene in E. coli and mammalian cells.
authors.affiliation: Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea, Seoul, Korea.
authors.forename: Goo-Young
authors.initials: GY
authors.lastname: Kim
authors.forename: Min-Kyung
authors.initials: MK
authors.lastname: Nam
authors.forename: Sang-Soo
authors.initials: SS
authors.lastname: Kim
authors.forename: Ho-Young
authors.initials: HY
authors.lastname: Kim
authors.forename: Sang-Kyu
authors.initials: SK
authors.lastname: Lee
authors.forename: Hyangshuk
authors.initials: H
authors.lastname: Rhim
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D000074923:High-Temperature Requirement A Serine Peptidase 2; D051379:Mice; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D000074923:High-Temperature Requirement A Serine Peptidase 2; C000615915:Htra2 protein, mouse