Abdullah N, Chase H A
Department of Chemical and Environmental Engineering, University Putra Malaysia, Serdang, Malaysia.
Biotechnol Bioeng. 2005 Nov 20;92(4):501-13. doi: 10.1002/bit.20633.
Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
在使用固定化金属亲和色谱（IMAC）进行扩张床纯化后，已采用酶法从组氨酸标记的蛋白质上切割C端或N端的多组氨酸标签。本研究评估了分别使用因子Xa和一种基因工程外肽酶二肽基氨基肽酶-1（DAPase-1）去除C端和N端多组氨酸标签的情况。使用了由带有C端或N端多组氨酸标签的麦芽糖结合蛋白（MBP）组成的模型蛋白。因子Xa成功消化了MBP-His(6)的六组氨酸标签，DAPase-1成功消化了HT15-MBP的N端多组氨酸标签。通过SDS-PAGE和抗组氨酸抗体的Western印迹判断，将MBP-His(6)转化为MBP所需的时间为l6小时。当使用减法IMAC纯化去标签化的蛋白时，产率适中，为71%，尽管总回收率较高，为95%。同样，当采用“柱上”标签消化进行消化时，产率为79%，回收率为97%。柱上标签消化是指从仍与IMAC柱结合的多组氨酸标记蛋白上切割组氨酸标签。DAPase-I系统对HT15-MBP（1mg/mL）的N端多组氨酸标签的消化效果优于因子Xa，产率和回收率分别更高，为99%和95%。DAPase-I系统的消化速度更快，5小时即可完成，而因子Xa则需要16小时。使用简单的减法IMAC柱程序从消化混合物中分离出去标签化的MBP蛋白时，去标签化的蛋白出现在流出液和洗涤组分中，而残留的二肽和DAPase-I（设计为带有多组氨酸尾巴）则被吸附到柱上。使用MonoS阳离子交换剂进行FPLC分析，以了解和监测DAPase-I将HT15-MBP消化为MBP的过程和时间进程。针对DAPase-I将HT15-MBP消化为MBP的系统，对温度、蛋白质浓度和酶活性等工艺变量进行了优化。简而言之，本研究证明，使用因子Xa或DAPase-I消化多组氨酸标签简单有效，且可在温和的反应条件下进行。
