Journal: PLoS ONE
Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
Figure Lengend Snippet: Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).
Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).
Techniques: Clone Assay, Expressing, Purification, Recombinant, Amplification, Plasmid Preparation, Transformation Assay, Sequencing, SDS Page, Affinity Chromatography, Incubation, Fast Protein Liquid Chromatography, Western Blot