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首页> 外文期刊>Biotechnology Progress >Genetic engineering strategies for purification of recombinant proteinsfrom Canola by anion exchange chromatography: An example ofbeta-glucuronidase
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Genetic engineering strategies for purification of recombinant proteinsfrom Canola by anion exchange chromatography: An example ofbeta-glucuronidase

机译：阴离子交换色谱法从油菜中纯化重组蛋白的基因工程策略：β-葡萄糖醛酸苷酶的实例

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The elution behavior of native canola proteins from different anion-exchange resins was determined. The elution profiles showed the potential for simplified recovery of acidic recombinant proteins from canola. When Q-sepharose fast flow was used, there were three optimal salt elution points at which a recombinant protein would have minimal contamination with native proteins. The feasibility of exploiting this advantage was examined for recovery of the acidic protein beta -glucuronidase (GUS/ GUSD0 from the Escherichia coli gene) along with three polyaspartate fusions to the wild-type GUS. The fusions contained 5 (GUSD5), 10 (GUSD10), or 15 (GUSD15) aspartic acids fused to the C-terminus and were chosen to extend the elution time. The three fusions and the wild-type enzyme were produced in E. coli, purified, and added to canola extracts before chromatography. The equivalence of this spiking experiment to that of extracting a recombinant protein from transgenic canola was determined in a control experiment using transgenic canola expressing the wild-type enzyme. Behavior in the transgenic and spiked experiments was equivalent. GUSD0 eluted at the earliest optimal elution point; the addition of polyaspartate tails resulted in longer retention times and better selective recovery. If one assumes binding through a single fusion (the protein is a tetramer), there is a nearly linear shift in elution within the salt gradient of 17 mM per added charge up to 10, with a reduced increment from 10 to 15. The fusions and their enzymatic activity proved very stable in the canola extracts through 7 days in cold storage, providing flexibility in process scheduling.
机译：测定了来自不同阴离子交换树脂的天然低芥酸菜子蛋白的洗脱行为。洗脱曲线显示了从油菜中简化回收酸性重组蛋白的潜力。当使用Q-琼脂糖快速流动时，存在三个最佳的盐洗脱点，在该点上重组蛋白对天然蛋白的污染极小。研究了利用这一优势的可行性，以回收酸性蛋白β-葡萄糖醛酸苷酶（来自大肠杆菌基因的GUS / GUSD0）以及与野生型GUS的三种聚天冬氨酸融合体。融合物包含5个（GUSD5），10（GUSD10）或15（GUSD15）与C末端融合的天冬氨酸，并被选择来延长洗脱时间。三种融合体和野生型酶均在大肠杆菌中产生，纯化，并在色谱分离前添加至油菜提取物中。在使用表达野生型酶的转基因油菜的对照实验中，确定了该加标实验与从转基因油菜中提取重组蛋白的当量。在转基因和加标实验中的行为是等效的。 GUSD0在最早的最佳洗脱点洗脱;聚天冬氨酸尾部的加入导致更长的保留时间和更好的选择性回收率。如果假设通过一次融合来结合（蛋白质是四聚体），则在每次添加电荷的17 mM盐梯度中，洗脱液几乎线性变化，最多10次，从10减少到15。他们的酶促活性在低温保存的菜籽油中经过7天的试验证明非常稳定，从而为工艺安排提供了灵活性。

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