Expression and purification of active recombinant equine lysozyme in Escherichia coli.
Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni(2+) affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.
Casaite
Vida
V
Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Mokslininku 12, Vilnius LT-08662, Lithuania. vidac@bchi.lt
Bruzyte
Simona
S
Bukauskas
Virginijus
V
Setkus
Arunas
A
Morozova-Roche
Ludmilla A
LA
Meskys
Rolandas
R
D000682:Amyloid / Q000378:metabolism; D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D006736:Horses; D009113:Muramidase / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism
D000682:Amyloid; D011994:Recombinant Proteins; D009113:Muramidase