Structural and functional characterization of ovotransferrin produced by Pichia pastoris.
Ovotransferrin is an egg white protein with complex disulfide and bilobal structures, which is derived from the same gene as chicken serum transferrin. We demonstrate here the structural and functional characteristics of bilobal ovotransferrin, produced at a high level using Pichia pastoris expression system. The recombinant protein was secreted into the medium, and the secretion signal peptide was processed correctly. The secretion level was almost 100 mg/l culture and the yield after purification by two-step anion exchange chromatography was 57 mg/l. The CD spectrum and fluorescence spectra indicate the correct folding of the recombinant protein. The analyses for the Fe3+ binding ability by urea-PAGE and visible absorption spectrum revealed that two Fe3+ sites exist in a recombinant ovotransferrin molecule as in the egg white protein. Endoglycosidases, such as endo-beta-N-acetylglucosaminidase H (Endo-H), peptide:N-glycosidase F (PNGaseF), and endo-beta-N-acetylglucosaminidase from Mucor hiemalis, showed differential activities for the native Fe3+-loaded, native Fe3+-free, and denatured forms of recombinant ovotransferrin; only the first enzyme displayed the cleavage ability for all the ovotransferrin forms. The results from the enzyme specificity and from the molecular weight difference for the intact and deglycosylated proteins were consistent with the view that recombinant ovotransferrin have one N-linked carbohydrate chain which mainly consists of two GlcNac and 10 mannoses.
The Graduate School of Agriculture, Kyoto University, Japan.
Kimihiko
K
Mizutani
Ikuko
I
Okamoto
Kiyotaka
K
Fujita
Kenji
K
Yamamoto
Masaaki
M
Hirose
D002241:Carbohydrates / Q000737:chemistry; D002942:Circular Dichroism; D003207:Conalbumin / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000737:chemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D033862:Iron-Binding Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008970:Molecular Weight; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D014170:Transformation, Genetic; D014508:Urea / Q000737:chemistry
D002241:Carbohydrates; D007202:Indicators and Reagents; D033862:Iron-Binding Proteins; D011994:Recombinant Proteins; D003207:Conalbumin; D014508:Urea; D006026:Glycoside Hydrolases