Department of Chemical and Natural Resources Engineering, Faculty of Engineering, Universiti Malaysia Pahang, 25000 Kuantan, Pahang, Malaysia.
J Chromatogr A. 2010 Feb 19;1217(8):1293-7. doi: 10.1016/j.chroma.2009.12.039. Epub 2009 Dec 22.
A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPas. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.
成功地开发了一种疏水相互作用扩展床吸附色谱（HI-EBAC），可直接从粗制大肠杆菌（E. coli）匀浆中回收尼帕病毒（NiV）的重组核衣壳蛋白（NCp-NiV）。与重组蛋白一起共释放的核酸增加了大肠杆菌匀浆的粘度，从而影响了 EBAC 柱中的轴向混合。因此，在将其加载到装有疏水相互作用色谱（HIC）吸附剂的 EBAC 柱之前，添加了 DNA 酶以降低原料的粘度。在洗涤缓冲液中添加甘油可减少洗涤缓冲液的体积，从而减少洗涤阶段 NCp-NiV 的损失。研究了流速、床膨胀度和流动相粘度对 HI-EBAC 吸附效率的影响。在 178 cm/h 的线性流速、2 倍的床膨胀度和 3.4 mPas 的流动相粘度下，达到了 3.2mg/g 吸附剂 10%穿透时的动态结合容量。使用含有盐浓度逐步梯度的缓冲液洗脱吸附的 NCp-NiV。使用 HI-EBAC 柱对疏水性 NCp-NiV 的纯化，从未澄清的大肠杆菌匀浆中回收了 80%的 NCp-NiV，纯化因子为 12.5。
