CN108239628A - 一种重组肠激酶的纯化方法 - Google Patents
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Abstract
本发明提供一种重组肠激酶的纯化方法,所述方法采用芳香族疏水层析填料,通过洗脱液洗脱得到纯品;所述洗脱液由洗脱缓冲液A与洗脱缓冲液B等体积混合形成,所述洗脱缓冲液A的配比为:20mmol Tris‑HCl、1mol/L NH4Cl,pH为7.0‑8.0;所述洗脱缓冲液B的配比为20mmolTris‑HCl,pH为7.0‑8.0。本发明提供的重组肠激酶的纯化方法,仅需要一次纯化洗脱就可以得到电泳纯的肠激酶样品,避免了多次反复纯化所带来的产品流失以及成本损耗,在得到纯品的同时收率得到大幅度的提高。
Description
技术领域
本发明涉及药物合成及化工领域,特别是涉及一种重组肠激酶的纯化方法。
背景技术
肠激酶(enterokinase)存在于高等动物的十二指肠粘膜中,是使胰蛋白酶原水解而成为活性胰蛋白酶的肽链内切(endo-peptidase)。此反应要比由胰蛋白酶自身触媒引起的活性化快数十倍,且其切割位点具有高度专一性,特异性识别DDDDK序列,关于肠激酶的应用研究、重组研究已经逐步被业内人士所重视。
目前,在肠激酶的纯化领域一般都是采用阴离子层析纯化、阳离子层析纯化、反相层析纯化交替进行的方式进行,为了获得纯度较高的肠激酶需要进行多次纯化,费时、费力,并且最终产率低,不符合工业化大量生产的需求。
肠激酶的纯化过程中,对于具有活性的粗酶液纯化,是整个制备过程的最后一个步骤,也是非常关键的步骤,如何获得高纯度的肠激酶同时收率提高对于重组肠激酶的制备来说至关重要,目前的纯化方法普遍存在的纯化难度高、纯化步骤复杂、纯化收率低等问题;此外,现有技术的纯化方法中,还存在洗脱液中含有影响肠激酶活性的乙醇、且存在分辨率低,成本高等缺点。
发明内容
针对上述现有技术存在的问题,本发明提供一种重组肠激酶的纯化方法,仅需要一次纯化洗脱就可以得到电泳纯的肠激酶样品,避免了多次反复纯化所带来的产品流失以及成本损耗,在得到纯品的同时收率得到大幅度的提高。
本发明所采用的技术方案为:一种重组肠激酶的纯化方法,所述方法采用芳香族疏水层析填料,通过洗脱液洗脱得到纯品;所述洗脱液由洗脱缓冲液A与洗脱缓冲液B等体积混合形成,所述洗脱缓冲液A的配比为:20mmol Tris-HCl、1mol/L NH4Cl,pH为7.0-8.0;所述洗脱缓冲液B的配比为20mmolTris-HCl,pH为7.0-8.0。
优选的,所述芳香族疏水层析填料中的疏水配基为苯基,如Phenyl 6FF疏水层析填料;疏水配基为苯基的填料,化学物理稳定性好,选择性高,高流速低反压,容易与蛋白质和多肽等生物大分子的表面的疏水性基团作用而结合,不同的分子疏水性不同,与疏水填料的疏水作用力强弱不同,从而分离纯化不同的分子,且填料使用寿命更长。
优选的,洗脱缓冲液A或洗脱缓冲液B的pH均优选为7.2-7.8,更优选为7.5。
优选的,洗脱用缓冲液流速为:线流速50-150cm/h,优选80-120 cm/h,更优选100cm/h。
优选的,所述芳香族疏水层析填料,首先在洗脱液中浸泡2-5h,浸泡后的填料装柱形成层析柱。
本发明相对于现有技术的优势:
1)本发明采用芳香族疏水层析填料,特别是疏水配基为苯基的填料,化学物理稳定性好,选择性高,高流速低反压,容易与蛋白质和多肽等生物大分子的表面的疏水性基团作用而结合,不同的分子疏水性不同,与疏水填料的疏水作用力强弱不同,从而分离纯化不同的分子,且填料使用寿命更长;
2)本发明洗脱液中不含有乙醇,不会影响肠激酶的活性,能够很好的保持肠激酶活性;
3)本发明的洗脱缓冲液A或洗脱缓冲液B中均含有NH4Cl,能增强疏水基团相互作用和结合,使疏水配基能够与蛋白之间相互作用,且梯度洗脱时,盐浓度逐渐降低,样品根据疏水性依次洗脱;
4)本发明发现pH为7.0-8.0条件下分辨率以及样品纯度会得到提高,尤其是在pH值为7.5时,分辨率以及样品纯度最高;
综上,本发明所述方法,相对于现有技术填料寿命长、分辨率以及样品纯度高,大大节约了成本。
附图说明
图1为层析谱图;
图2为不同洗脱峰的电泳分析图;
其中:1酶解混合液,2洗脱峰1,3洗脱峰2,4洗脱峰3,5 Marker。
具体实施方式
为进一步理解本发明,下面结合实施对本发明所公开的纯化肠激酶的方法进行详细说明,本发明的保护范围不受以下实施例的限制
以下实施例中所述实验方法,如无特殊说明,均为常规方法,所涉及试剂和材料,如无特殊说明,均为商业途径的市售产品。
本发明所用的用于纯化的粗酶液可以为按照任何现有技术中的相关内容获得包含有肠激酶的混合液;举例但不限于下述方法:
参照专利CN 105802944 A 发酵结束后,离心收集菌体,按质量体积比1∶10加入破碎缓冲液(25mmol/LTris-HCL+5mmol/LEDTA,,PH7.5),高压均质机700bar条件下破碎两遍,离心收集包涵体沉淀,包涵体湿重为30g/L发酵液。将沉淀按重量体积比1∶10加入洗涤缓冲液(2mol/L尿素+1.5%Triton),室温磁力搅拌1小时,离心收集的沉淀用洗涤缓冲液洗涤两次。再用包涵体溶解缓冲液(8mol/L尿素+10mmol/LEDTA+25mmol/LTris-HCL,PH7.5)按重量体积比1∶10溶解过夜。溶解后的包涵体经离心、超滤去除杂质后,稀释到蛋白浓度0.3 mg/ml,在复性缓冲液(0.2mol/L尿素+10mmol/LEDTA+25mmol/LTris-HCL,GSH:GSSG=1mmol/L:0.3mmol/L,PH10)中,7-10℃复性过夜,复性后的肠激酶原蛋白经肠激酶(1:200)在室温下酶解2小时后即可得到具有活性的待分离的肠激酶粗酶液。
实施例1
1.1洗脱液准备
缓冲液A的制备:20mmolTris-HCl和1M NH4Cl缓冲液形成的pH为7.5的缓冲液;
缓冲液B的制备:20mmolTris-HCl,形成pH为7.5的缓冲液;
将缓冲液A和缓冲液B等体积混合后,形成洗脱用洗脱液。
1.2层析柱准备
取Phenyl 6FF(Phenyl Bestarose 6 Fast Flow)疏水层析填料,在1.1中配置的pH7.5的洗脱液中浸泡2-5h,将浸泡处理后的填料装柱,形成待用的Phenyl 6FF疏水层析柱。
1.3层析纯化
层析设备为:AKTA中低压层析系统,洗脱用缓冲液流速为:线流速100cm/h,分别收集不同洗脱峰处的洗脱液,得到的层析图谱如图1所示,对洗脱液进行电泳分析,分析结果如图2所示。通过结果可以看出,通过一次洗脱就可以将肠激酶纯化,并得到电泳纯的肠激酶。
对比例1
将肠激酶分离液在pH8.5的UniPS30-300层析柱中通过等体积混合的洗脱缓冲液A与洗脱缓冲液B洗脱得到,所述洗脱缓冲液A是由10%乙醇与5mmol Tris-HCl缓冲液形成的pH为8.5的缓冲液,洗脱缓冲液B是由50%乙醇与5mmol Tris-HCl缓冲液形成的pH为8.5的缓冲液。
实施例1与与对比例1的实验数据对比:
填料名称 | 上样载量(mg/ml) | 收率% | 纯度 |
对比例1 | 3 | 25 | 电泳纯 |
实施例1 | 10 | 70 | 电泳纯 |
实施例2
1.1洗脱液准备
缓冲液A的制备:20mmolTris-HCl和1M NH4Cl缓冲液形成的pH为7.0的缓冲液;
缓冲液B的制备:20mmolTris-HCl,形成pH为8.0的缓冲液;
将缓冲液A和缓冲液B等体积混合后,形成洗脱用洗脱液。
1.2层析柱准备
取Phenyl 6FF(Phenyl Bestarose 6 Fast Flow)疏水层析填料,在1.1中配置的洗脱液中浸泡2-5h,将处理后的填料装柱,形成待用的Phenyl 6FF疏水层析柱。
1.3层析纯化
层析设备为:AKTA中低压层析系统,洗脱用缓冲液流速为:线流速80cm/h,分别收集不同洗脱峰处的洗脱液,对洗脱液进行电泳分析。结果显示,通过一次洗脱就可以将肠激酶纯化,并得到电泳纯的肠激酶。
实施例3
1.1洗脱液准备
缓冲液A的制备:20mmolTris-HCl和1M NH4Cl缓冲液形成的pH为8.0的缓冲液;
缓冲液B的制备:20mmolTris-HCl,形成pH为7.0的缓冲液;
将缓冲液A和缓冲液B等体积混合后,形成洗脱用洗脱液。
1.2层析柱准备
取Phenyl 6FF(Phenyl Bestarose 6 Fast Flow)疏水层析填料,在1.1中配置的洗脱液中浸泡2-5h,将处理后的填料装柱,形成待用的Phenyl 6FF疏水层析柱。
1.3层析纯化
层析设备为:AKTA中低压层析系统,洗脱用缓冲液流速为:线流速120cm/h,分别收集不同洗脱峰处的洗脱液,对洗脱液进行电泳分析。结果显示,通过一次洗脱就可以将肠激酶纯化,并得到电泳纯的肠激酶。
Claims (8)
1.一种重组肠激酶的纯化方法,其特征在于,所述方法采用芳香族疏水层析填料,通过洗脱液洗脱得到纯品;所述洗脱液由洗脱缓冲液A与洗脱缓冲液B等体积混合形成,所述洗脱缓冲液A的配比为:20mmol Tris-HCl、1mol/L NH4Cl,pH为7.0-8.0;所述洗脱缓冲液B的配比为20mmolTris-HCl,pH为7.0-8.0。
2.根据权利要求1所述的重组肠激酶的纯化方法,其特征在于,所述芳香族疏水层析填料中的疏水配基为苯基。
3.根据权利要求2所述的重组肠激酶的纯化方法,其特征在于,洗脱缓冲液A或洗脱缓冲液B的pH为7.2-7.8。
4.根据权利要求3所述的重组肠激酶的纯化方法,其特征在于,洗脱缓冲液A或洗脱缓冲液B的pH为均7.5。
5.根据权利要求1所述的重组肠激酶的纯化方法,其特征在于,洗脱用缓冲液流速为:线流速50-150cm/h。
6.根据权利要求5所述的重组肠激酶的纯化方法,其特征在于,洗脱用缓冲液流速为:线流速80-120 cm/h。
7.根据权利要求6所述的重组肠激酶的纯化方法,其特征在于,洗脱用缓冲液流速为:线流速100cm/h。
8.根据权利要求1所述的重组肠激酶的纯化方法,其特征在于,所述芳香族疏水层析填料,首先在洗脱液中浸泡2-5h,浸泡后的填料装柱形成层析柱。
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