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Expression and Purification of Goat Lactoferrin from Pichia pastoris Expression System

Authors

  • Gen-Hung Chen,

    1. Author Chen is with Dept. of Cosmetic Science, Providence Univ., 200, Chung-Chi Rd., Taichung 43301, Taiwan. Author Yin is with Dept. of Sea Food Science, Natl. Kaohsiung Marine Univ., 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung, Taiwan. Authors Chiang and Jiang are with Dept. of Food & Nutrition, Providence Univ. Author Jiang is with Dept. of Food Science, Natl. Taiwan Ocean Univ., Keelung 202, Taiwan. Direct inquiries to author Jiang (E-mail: stjiang@pu.edu.tw).
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  • Li-Jung Yin,

    1. Author Chen is with Dept. of Cosmetic Science, Providence Univ., 200, Chung-Chi Rd., Taichung 43301, Taiwan. Author Yin is with Dept. of Sea Food Science, Natl. Kaohsiung Marine Univ., 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung, Taiwan. Authors Chiang and Jiang are with Dept. of Food & Nutrition, Providence Univ. Author Jiang is with Dept. of Food Science, Natl. Taiwan Ocean Univ., Keelung 202, Taiwan. Direct inquiries to author Jiang (E-mail: stjiang@pu.edu.tw).
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  • I-Hua Chiang,

    1. Author Chen is with Dept. of Cosmetic Science, Providence Univ., 200, Chung-Chi Rd., Taichung 43301, Taiwan. Author Yin is with Dept. of Sea Food Science, Natl. Kaohsiung Marine Univ., 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung, Taiwan. Authors Chiang and Jiang are with Dept. of Food & Nutrition, Providence Univ. Author Jiang is with Dept. of Food Science, Natl. Taiwan Ocean Univ., Keelung 202, Taiwan. Direct inquiries to author Jiang (E-mail: stjiang@pu.edu.tw).
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  • Shann-Tzong Jiang

    1. Author Chen is with Dept. of Cosmetic Science, Providence Univ., 200, Chung-Chi Rd., Taichung 43301, Taiwan. Author Yin is with Dept. of Sea Food Science, Natl. Kaohsiung Marine Univ., 142, Hai-Chuan Rd. Nan-Tzu, Kaohsiung, Taiwan. Authors Chiang and Jiang are with Dept. of Food & Nutrition, Providence Univ. Author Jiang is with Dept. of Food Science, Natl. Taiwan Ocean Univ., Keelung 202, Taiwan. Direct inquiries to author Jiang (E-mail: stjiang@pu.edu.tw).
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Abstract

ABSTRACT:  The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZαC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZαC into Pichia pastoris X-33 expression host, the GLF-pGAPZαC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using α-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.

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