S_1125_9_TAGZyme_DAPase_Enzyme
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TAGZyme DAPase Enzyme (2.5 U)

Cat. No. / ID:   34362

用于处理大约 50 mg 标签蛋白:2.5 单位 DAPase 酶,20 mM 盐酸半胱胺 (1 mL)
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EnzymeVector
TAGZyme Enzyme
TAGZyme pQE Vector
Quantity
2.5 U (1 mL)
50 U (25 mL)
该产品将于 September 30, 2025 起停产,或存货耗尽时停售。
TAGZyme System 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。
是否需要商用批量、定制或优化产品?我们还提供物流、合规等方面的支持。主动联系,与 QIAGEN 战略合作伙伴及 OEM 合作

特点

  • 经过优化,通过 TAGZyme 酶去除表达的 His 标签
  • 高效去除 His 标签:在 37°C 下只需 30 分钟即可去除 >95%
  • N-末端 His 标签蛋白的高水平表达
  • 高纯度终产物
  • 通过 Ni-NTA 法完全去除污染物

产品详情

TAGZyme Enzyme DAPase 包含足够从多达 10 mg His 标签蛋白中高度特异性和高效去除 His 标签的酶。TAGZyme 系统可用于从使用 TAGZyme pQE-2 载体表达的含有内在 DAPase 停止点的蛋白质中去除 His 标签。

绩效

TAGZyme DAPase Enzyme 可高效地从 N-末端 His 标签起顺序去除二肽,直到使用 TAGZyme pQE-2 载体表达的“停止点”。

原理

His 标签重组蛋白已成为研究蛋白质结构和功能的宝贵工具。His 标签的小尺寸和低免疫原性意味着通常不需要去除它。然而,对于某些应用而言,例如通过 X 线或 NMR 进行的结构测定研究或治疗蛋白的生产,最好使用不含载体衍生氨基酸的蛋白产物。

TAGZyme pQE-2 载体适用于含有内在 DAPase 终止位点的蛋白质。 

TAGZyme 系统能够以高度特异性和高效率从重组蛋白中去除 N-末端 His 标签。DAPase 酶用于从经过纯化的 His 标签蛋白的 N-末端顺序剪切二肽(参见图片 “His 标签去除”)。这种酶切在酶到达“停止点”时停止,后者是一种不能作为底物的氨基酸基序(参见表格“DAPase 停止点”)。

DAPase 终止位点

氨基酸 DAPase 停止点 (↓) 序列*
赖氨酸 (Lys, K) Xaa-Xaa...Xaa-Xaa ↓ Lys-Xaa...
精氨酸 (Arg, R) Xaa-Xaa...Xaa-Xaa ↓ Arg-Xaa...
脯氨酸 (Pro, P) Xaa-Xaa...Xaa-Xaa ↓ Xaa-Xaa-Pro-Xaa...
脯氨酸 (Pro, P) Xaa-Xaa...Xaa-Xaa ↓ Xaa-Pro-Xaa-Xaa...
谷氨酰胺 (Gln, Q)† Xaa-Xaa...Xaa-Xaa ↓ Gln-Xaa...
异亮氨酸 (Ile, I) Xaa-Xaa...Xaa-Xaa ↓ Xaa-Ile-Xaa-Xaa...
查看图表

程序

对于含有内在停止点的重组蛋白,使用 TAGZyme pQE-2 载体表达能够完全、高效地去除 N-末端 His 标签,而与 DNA 插入片段的克隆位点无关(参见图片 “His 标签去除”)。与 DAPase 酶一起孵育后,使用 Ni-NTA 基质对反应混合物进行消减固定化金属亲和层析 (immobilized-metal affinity chromatography, IMAC)(参见图片 “去标签蛋白的纯化”)。His 标签片段及 TAGZyme DAPase Enzyme(携带 C-末端 6xHis 标签)与基质结合,并且在流穿液中回收得到纯的去标签目标蛋白。

查看图表

应用

TAGZyme 系统提供了特异性剪切、重组试剂使用以及所有污染物的完全去除,使其成为生产无 His 标签蛋白的首选方法,其应用包括:

  • 通过 NMR 或 X 线衍射晶体分析法进行蛋白质结构测定
  • 治疗蛋白的生产

辅助数据和图表

资源

产品选择指南 (1)
载体序列 (2)
For the pQE-2 vector
For the pQE-1 vector
Package Insert (1)
安全数据表 (1)
Download Safety Data Sheets for QIAGEN product components.
试剂盒操作手册 (1)
TAGZyme Handbook
PDF (2MB)
For exoproteolytic cleavage of N-terminal His tags
Safety Data Sheets (1)
Certificates of Analysis (1)

Publications

Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy.
Block H; Kubicek J; Labahn J; Roth U; Schäfer F;
Protein Expr Purif; 2007; 57 (2):244-54 2007 Oct 17 PMID:18053740

FAQ

Is it possible to cleave the 6xHis-tag from an expressed protein?

Yes. In order to cleave off an N-terminal 6xHis tag, a protease cleavage site must be inserted between the coding sequences of the tag and the N-terminus of the protein. Factor Xa Protease recognizes the amino acid sequence Ile-Glu-Gly-Arg and cleaves the peptide bond C-terminal of the arginine residue. The expression vector pQE-30 Xa encodes a Factor Xa Protease recognition site between the N-terminal 6xHis-tag sequence and the multiple cloning site.

If the gene of interest is cloned blunt ended at the 5´-end using the StuI restriction site of the vector, Factor Xa Protease cleavage of the purified recombinant protein results in a protein product without any vector-derived amino acids at the N-terminus.

Tags can also be removed exoproteolytically using the TAGZyme System. This system is an efficient and specific solution for the complete removal of small N-terminal His tags and other amino acid tags by the use of exopeptidases. For detailed information on the procedure please review the TAGZyme handbook.

Please note that both tag removal options work on N-terminal 6xHis tags only.

 

 

FAQ ID -140
Does endogenously expressed DAPase in human cells degrade proteins that are expressed in cell culture?
DAPase (dipeptidyl aminopeptidase I = Cathepsin C), even though endogenously expressed in human tissues and cells, will have only a negligible effect on the degradation of proteins expressed in cell culture. The reason for this is the low level of endogenous DAPase compared to the much higher level of typically overexpressed recombinant protein. However, it is always worthwhile to run a time-course expression experiment to check for possible protein degradation, since proteases and peptidases tend to degrade proteins over time.
FAQ ID -527
How complete is the removal of DAPase in the TAGZyme process?
The removal of DAPase is >99%.
FAQ ID -319
What are the calculated molecular weights of the TAGZyme enzymes?
DAPase: heterodimer, 24 kDa and 6 kDa subunit; Qcyclase: 35 kDa; pGAPase: 28 kDa
FAQ ID -329
Is it possible to store TAGzyme enzymes at°C?
Yes. The TAGzyme enzymes do not experience any loss in function after several freeze and thaw cycles. However, storage at -80°C is not necessary as storage at -20°C is adequate.
-80