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首页> 外文期刊>Biotechnology Progress >Preparative purification of a recombinant protein by hydrophobic interaction chromatography:modulation of selectivity by the use of chaotropic additives
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Preparative purification of a recombinant protein by hydrophobic interaction chromatography:modulation of selectivity by the use of chaotropic additives

机译：通过疏水相互作用色谱法制备重组蛋白：使用离液添加剂调节选择性

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摘要

Development and implementation of a chaotropic wash step following protein loading on a hydrophobic interaction chromatographic (HIC)column is described for the purification of a recombinant protein.Various agents that reduce protein affinity in hydrophobic interaction chromatographic systems were screened for their utility in a wash step follwing protein loading on a Phenyl Fast Flow Sepharose HIC column.A combination of sodium thiocyanate,glycerol,and urea was selected as a suitable additive for the wash buffer that selectivley eluted msot of the major impurities present in the feed stream.Eluate purity,as monitored by reversed-phase chromatography and SDS-PAGE,was significnalty increased by incorporation of this wash step in the purification process.Incorporation of this wash step on HIC enabled a reduction in the overall number of chromatographic steps in the down stream purification process for this recombinant protein,resulting in improved process yields and significant economic advantages.The effect of varying concentrations of each of the three wash additives on yield was studied.While the step yield decreased with an increase in concentration for urea and sodium thiocyanate,an optimum was observed with respect to glycerol concentration.The preferential interaction theory is employed to explain this effect.
机译：描述了蛋白质在疏水相互作用色谱（HIC）柱上加载后离液序列高的洗涤步骤的开发和实施，用于纯化重组蛋白。筛选了在疏水相互作用色谱系统中降低蛋白质亲和力的各种试剂在洗涤步骤中的效用将蛋白质加载到苯基快速流动琼脂糖凝胶HIC柱上。选择硫氰酸钠，甘油和尿素的组合作为洗涤缓冲液的合适添加剂，以选择性洗脱进料流中存在的主要杂质。洗脱纯度通过在纯化过程中加入此洗涤步骤可提高通过反相色谱和SDS-PAGE监测的显着性。在HIC上加入此洗涤步骤可减少下游纯化过程中色谱步骤的总数重组蛋白，可提高工艺产量并显着节约经济研究了三种洗涤添加剂的不同浓度对收率的影响。虽然随着尿素和硫氰酸钠浓度的增加分步收率降低，但相对于甘油浓度却观察到了最佳值。优先相互作用理论是用来解释这种效果。

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