Ahn Byung Cheol, Park Jeong Soo, Kim Dongjun, Park Junho, Pi Jia, Yum Jung Sun, Jeong Yongsu, Baek Kwanghee, Moon Hong Mo, Yoon Jaeseung
R&D Center, CHA Vaccine Institute, Seongnam-si, Republic of Korea.
Protein Expr Purif. 2013 Mar;88(1):1-6. doi: 10.1016/j.pep.2012.11.007. Epub 2012 Nov 29.
Mannose-binding lectin (MBL) is an important serum protein that functions in the innate immune system and has been considered to have therapeutic potential in MBL replacement therapies for patients with deficient or low levels of MBL. In this study, we established a Chinese hamster ovary (CHO) cell line that overexpresses the recombinant human MBL (rhMBL) protein. In an 11-day batch culture process using a 30-L bioreactor (20-L working volume) and serum-free medium, these cells could produce over 226 mg/L of rhMBL protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a three-step chromatographic procedure that resulted in a recovery rate of approximately 55%. This purified rhMBL protein adopted oligomeric bouquet-like structures that were similar to those of native MBL present in human blood, and these oligomeric structures were reported to be critical in MBL functions. We further demonstrated in carbohydrate binding and complementation activation assays that this rhMBL protein was functionally active with very similar dissociation constants and half maximal effective concentrations to those of native MBL.
甘露糖结合凝集素（MBL）是一种重要的血清蛋白，在先天免疫系统中发挥作用，并且在针对MBL缺乏或水平低下患者的MBL替代疗法中被认为具有治疗潜力。在本研究中，我们建立了一种过表达重组人MBL（rhMBL）蛋白的中国仓鼠卵巢（CHO）细胞系。在使用30升生物反应器（工作体积20升）和无血清培养基的11天分批培养过程中，这些细胞能够产生超过226毫克/升的rhMBL蛋白。然后使用三步色谱法从培养上清液中纯化重组蛋白至同质，回收率约为55%。这种纯化的rhMBL蛋白呈现出类似于人血液中天然MBL的寡聚花束状结构，据报道这些寡聚结构对MBL功能至关重要。我们进一步在碳水化合物结合和补体激活试验中证明，这种rhMBL蛋白具有功能活性，其解离常数和半数最大效应浓度与天然MBL非常相似。
