title: Expression and purification of goat lactoferrin from Pichia pastoris expression system.
abstract: The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZalphaC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZalphaC into Pichia pastoris X-33 expression host, the GLF-pGAPZalphaC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using alpha-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.
authors.affiliation: Dept. of Cosmetic Science, Providence Univ., 200, Chung-Chi Rd., Taichung 43301, Taiwan.
authors.forename: Gen-Hung
authors.initials: GH
authors.lastname: Chen
authors.forename: Li-Jung
authors.initials: LJ
authors.lastname: Yin
authors.forename: I-Hua
authors.initials: IH
authors.lastname: Chiang
authors.forename: Shann-Tzong
authors.initials: ST
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006031:Glycosylation; D006041:Goats / Q000235:genetics; D007501:Iron / Q000378:metabolism*; D007781:Lactoferrin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins; D020539:Sequence Analysis, Protein
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D007501:Iron; D007781:Lactoferrin