融合标签去除酶系列：去除蛋白质标签的有效手段

 

为了提升重组蛋白的产量或便于纯化或检测等，在克隆过程中，常常在目标蛋白的N端或C端添加额外的标签。这些标签依据其功能分为两类：纯化标签和可溶性标签。纯化标签有助于通过亲和层析快速提取蛋白；而可溶性标签则有助于蛋白质的正确折叠和提高其溶解性。

 

常见的蛋白质标签包括6*His、GST、MBP、StrepⅡ、Flag、SUMO以及复合型双标签或多标签。由于GST、MBP、SUMO等蛋白标签的分子量较大，它们通常需要被移除，以避免对目标蛋白的结构和功能造成影响。在结构生物学或其他高精度研究中，即使是小分子量的多肽标签（例如StrepⅡ、Flag），也常常被去除以获得最佳结果。在实际操作中，化学切割方法较少被采用，因为它的裂解位点特异性不高，且可能会对目标蛋白造成不必要的修饰。目前，更倾向于使用反应条件温和且具有高度位点特异性的酶解法。

 

活性数据：

Enterokinase, Bovine

The substrate of 50 μ g fusion protein was digested by enzyme, and the addition amount of enterokinase in the sample was 0.5IU, 1IU, 1.5IU and 2IU, respectively. The substrate is a fusion protein with a molecular weight of 10 KD and reacted at 25 ℃ for 16 hours, and the target band of 8.4kD is produced after restriction enzyme digestion.

Recombinant Enterokinase (High specific activity)

The results of 500ug substrate digestion separated under different quantity of hEK, the reaction was incubated for 16 h at 4°C.

M marker

Lane 1 Substrate/hEK=50/1

Lane 2 Substrate/ hEK =100/1

Lane 3 Substrate/ hEK =300/1

Lane 4 Substrate/ hEK =500/1（1U hEK）

Lane 5 Substrate/ hEK =700/1

Lane 6 Substrate/ hEK =1000/1

Lane 7 Substrate/ hEK =3000/1

Lane 8 Only substrate

 

Recombinant Tobacco Etch Virus Protease (rTEV)

The substrate of 3 μg fusion protein was digested by enzyme, and the addition amount of rTEV Protease in the sample was 0.3U, 0.6U, 1.0U, 1.3U and 2U, respectively. The substrate is a fusion protein with a molecular weight of 27 KD and reacted at 30 ℃ for 1 hour, and the target band of 18kD is produced after rTEV Protease digestion.

Recombinant SUMO Protease, Yeast

The substrate of 2 μg fusion protein was digested by SUMO Protease at 30 ℃ for 1 hour.

The addition amount of SUMO Protease were 0U, 0.25U, 0.5U, 0.75U and 1U, respectively.

The substrate is a fusion protein with a molecular weight of 20 KD and digested completely into two bands.

 

 HRV 3C Protease

The results of 100μg substrate digestion separated under different quantity of 3C Protease, the reaction was incubated for 16h minutes at 5°C.

M marker

Lane 1 Substrate/3C Protease=3000/1

Lane 2 Substrate/3C Protease=2500/1

Lane 3 Substrate/3C Protease=2000/1

Lane 4 Substrate/3C Protease=1000/1

Lane 5 Substrate/3C Protease=800/1

Lane 6 Substrate/3C Protease=600/1

Lane 7 Substrate/3C Protease=500/1

Lane 8 Substrate/3C Protease=300/1

Lane 9 Substrate/3C Protease=200/1

Lane 10 Substrate/3C Protease=100/1