Chen Gen-Hung, Yin Li-Jung, Chiang I-Hua, Jiang Shann-Tzong
Dept. of Cosmetic Science, Providence Univ., 200, Chung-Chi Rd., Taichung 43301, Taiwan.
J Food Sci. 2007 Mar;72(2):M67-71. doi: 10.1111/j.1750-3841.2007.00281.x.
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.
重组山羊乳铁蛋白（rGLF）是利用pGAPZalphaC载体、以GAP作为启动子、以博来霉素作为选择标记，在甲基营养型酵母毕赤酵母中表达的。将GLF-pGAPZalphaC转化到毕赤酵母X-33表达宿主中后，GLF-pGAPZalphaC载体整合到了毕赤酵母X-33染色体的GAP启动子位点。利用α-因子前导序列，rGLF得以表达并分泌到培养液中。SDS-PAGE和PAS染色分析表明，rGLF可通过肝素-琼脂糖6快速流动亲和层析纯化至电泳纯，并由表达宿主进行糖基化修饰。纯化后的rGLF产量约为每升培养液2.0毫克。其N端序列与天然山羊乳铁蛋白（nGLF）相同。纯化后的rGLF的铁结合行为、木瓜蛋白酶抑制特性和热稳定性与nGLF相当。这是利用毕赤酵母系统完整表达山羊乳铁蛋白的首次报道。
