乳腺生物反應器表達的重組人乳鐵蛋白的分離純化及生物活性鑒定

白倩，張焱，汪音爵，羅堅，李巖，黃永東，馬潤宇，蘇志國

1 北京化工大學生命科學與技術學院，北京 100029

2 中國科學院過程工程研究所 生化工程國家重點實驗室，北京 100190

生物技術與方法

乳腺生物反應器表達的重組人乳鐵蛋白的分離純化及生物活性鑒定

白倩1,2，張焱2，汪音爵2，羅堅2，李巖2，黃永東2，馬潤宇1，蘇志國2

1 北京化工大學生命科學與技術學院，北京 100029

2 中國科學院過程工程研究所 生化工程國家重點實驗室，北京 100190

以國產高交聯度的快流速瓊脂糖為基質，合成了不同配基密度的 SP (Sulfopropyl，磺酸基) 離子交換介質，建立了乳腺生物反應器表達重組人乳鐵蛋白 (Recombinant Human Lactoferrin，rHLF) 的純化方法。以溶菌酶為模型蛋白考察了不同配基密度離子交換介質的靜態和動態吸附行為，結果表明介質具有良好的吸附性能。不同配基密度離子交換介質均可純化得到rHLF，其中，高配基密度 (0.24 mol/L) 的離子交換介質每毫升可以處理50 mL rHLF牛乳，rHLF收率為86.5%，純度為98.5%。圓二色譜的測定結果表明純化的rHLF二級結構與天然人乳鐵蛋白一致。生物學功能實驗結果表明，rHLF的鐵結合與釋放活性與天然人乳鐵蛋白相似，濃度為5 g/L的rHLF對大腸桿菌的生長具有明顯的抑制作用。

離子交換介質，配基密度，重組人乳鐵蛋白，純化，抑菌活性

Abstract:Novel ion exchange adsorbents were synthesized by immobilizing sulfopropyl derivative onto homemade highly cross-linked agarose beads. The effects of different ligand densities (from 0.05 to 0.24 mol/L) on static and dynamic adsorption of the adsorbents were investigated using lysozyme as a model protein. Based on these results, rHLF was purified from the transgenic milk by our SP media. 1 mL high density (0.24 mol/L) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF. 5 g/L concentration of rHLF significantly inhibited the growth ofEscherichia coli. These studies lay a solid foundation for the wideapplication of our self-prepared ion exchange adsorbents in protein purification.

Keywords:ion exchange adsorbents, ligand density, recombinant human lactoferrin, purification, antimicrobial activity

乳鐵蛋白 (Lactoferrin，LF) 又名乳轉鐵蛋白，是轉鐵蛋白家族的重要一員。其分子量大約80 kDa[1]，等電點為8.7左右[2]。乳鐵蛋白分子中結合了兩條糖鏈，占分子量的7%左右。蛋白分子N端和C端的特征環狀結構可以結合三價鐵離子。乳鐵蛋白具有多種重要的生理功能，主要包括鐵吸收與釋放、抗菌活性以及炎癥反應中的鐵離子代謝等。……