title: Construction, non-denaturing affinity purification, and characterization of baculovirally expressed human secretory leukocyte protease inhibitor.
abstract: Secretory leukocyte protease inhibitor (SLPI) is a 11.7 kDa mucosal protein with potent anti-microbial, anti-inflammatory, and wound healing activities. Previous efforts to express and purify the non-glycosylated cationic protein as a recombinant protein in bacteria required extensive denaturation and renaturation to refold the disulfide-rich protein into its biologically active form. To overcome this limitation, we have expressed human SLPI as a polyhistidine-tagged protein (bvHisSLPI) using a recombinant baculovirus expression system. Studies were conducted to determine the timing of maximal protein production following baculovirus infection of Sf21 cells. The 16.4kDa-tagged protein was then overexpressed in Sf21 cells following a 48-h infection with bvHisSLPI-encoding baculovirus, purified by nickel-chelating affinity chromatography under non-denaturing conditions, and analyzed by Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Purified bvHisSLPI was further characterized by enterokinase digestion to remove the polyhistidine tag from its N-terminus. In serine protease inhibition assays, purified bvHisSLPI blocked substrate cleavage by two serine proteases, chymotrypsin and cathepsin G, comparable to bacterially expressed SLPI. The baculovirus expression and affinity purification strategy described here will facilitate further studies of the structural and biological properties of this important multifunctional protein.
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods*; D004765:Enteropeptidase / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D053481:Secretory Leukocyte Peptidase Inhibitor; D018411:Spodoptera / Q000166:cytology / Q000821:virology
chemical_list: D011480:Protease Inhibitors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D004765:Enteropeptidase