Affinity Purification Of Lactoferrin By Synthetic Ligand

Human lactoferrin is a 70~80 kDa iron‐binding glycoprotein of the transferring family with antibacterial, antifungal, antiviral, antitumor and immunomodulating activities. Owing to its implications in these functions, human lactoferrin has been considered a wide variety of applications in food and human health care, but its applications are restricted because of the limited source. Recently, a cattle mammary gland bioreactor expressing recombinant human lactoferrin has been generated by a research group led by Dr. Li Ning at Chinese Agricultural University, the level of recombinant human lactoferrin expressed by transgenic cattle could reach as high as 2.529±0.212 mg/mL～3.429±0.417 mg/mL, which probably provided a powerful approach to achieve large-scale production of recombinant human lactoferin in the milk of transgenic cows. The purpose of this study is to establish a novel affinity protocol for large-scale purification of recombinant human lactoferrin from the milk of transgenic cows by synthetic ligands.Immobilizedβ-Alanine, a synthetic ligand with high affinity for recombinant human lactoferrin was selected based on screening of a 322-member synthetic ligand library. By further study, Tris·HCl buffer(50 mmol/L, pH 7.0, 0.15 mol/L NaCl) and Gly·HCl buffer(0.1 mol/L, pH 2.4) were chosen as equilibration buffer and elution buffer respectively for this affinity protocol. By one-step purification with immobilizedβ-Alanine, the bound recombinant human lactoferrin was eluted with purity>95%, and the recovery could reach 81.6%. The maximal binding capacity of immobilizedβ-Alanine for recombinant human lactoferrin is as high as 65.5 mg/ g dry medium, moreover, this synthetic ligand could withstand the harsh cleaning and regeneration by NaOH solution.MALDI-TOF analysis identified that the purified protein was human lactoferrin, and the N-terminal sequencing further revealed that the N-terminal sequence of recombinant human lactoferrin was identical to that of native human lactoferrin, i.e., GRRRRSVQW.Tow most prominent biological activities of human lactoferrin, that is, iron-binding and–releasing properties and antibacterial activity, were investigated, and the results indicated that the biological activities of recombinant human lactoferrin were similar to that of native human lactoferrin. Both the recombinant human lactoferrin and native human lactoferrin could bind iron after treatment with Fe-NTA solution and release iron when pH was low. The iron began to be released around pH 4.5 and completely finished around pH 2.0. The growth of E.coli. was significantly inhibited when 5 mg/mL recombinant human lactoferrin was added to bacterial medium, which revealed that the recombinant human lactoferrin retained its natural biological activity after undergoing purifying procedures. In additional, immobilizedβ-Alanine could purify native human lactoferrin, bovine lactoferrin and goat lactoferrin from human milk, bovine milk and goat milk respectively, which predicts a vast potential for multiple applications of this ligand.In conclusion, immobilizedβ-Alanine can be applied to large-scale purification of recombinant human lactoferrin as well as native human lactoferin, bovine lactoferrin and goat lactoferrin respectively and has great potential for commercial application.