Expression and purification of goat lactoferrin from Pichia pastoris expression system.
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.
Chen
Gen-Hung
GH
Dept. of Cosmetic Science, Providence Univ., 200, Chung-Chi Rd., Taichung 43301, Taiwan.
Yin
Li-Jung
LJ
Chiang
I-Hua
IH
Jiang
Shann-Tzong
ST
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
D018076:DNA, Complementary; D011994:Recombinant Proteins; D007501:Iron; D007781:Lactoferrin