CN113214369A - 一种分子肽突变体 - Google Patents
一种分子肽突变体 Download PDFInfo
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- CN113214369A CN113214369A CN202110516304.3A CN202110516304A CN113214369A CN 113214369 A CN113214369 A CN 113214369A CN 202110516304 A CN202110516304 A CN 202110516304A CN 113214369 A CN113214369 A CN 113214369A
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Abstract
本发明公开一种分子肽突变体,氨基酸序列如SEQ ID NO:1所示。本发明在不影响异肽键形成的基础上对SpyCatcher进行设计改造得到对pH有刺激响应的分子肽SpyCatcher‑21,并且通过对晶体结构的分析在SpyCatcher‑21的关键loop中引入Pro来降低loop的柔性,获得突变体SpyCatcher‑21_A82P,提高了连接效率,可用于根据客观需要,通过改变环境的pH来得到不同程度的偶联来得到双酶催化;或与带有正电的酶通过静电作用相互作用得到三酶偶联的催化体系。
Description
技术领域
本发明属于分子肽SpyCatcher设计领域,更具体地,涉及一种分子肽突变体。
背景技术
在2010年,英国牛津大学生物化学中心Mark Howarth团队在革兰氏阳性菌酿脓链球菌(Streptococcus pyogenes)的菌毛蛋白中分离了可以自发形成异肽键的多肽片段,分别称为SpyTag(13个氨基酸)和SpyCatcher(116个氨基酸),其中SpyTag的Asp117可以和SpyCatcher的Lys31可以自发地脱水形成异肽键。SpyTag/SpyCatcher已经倍广泛的应用在各种领域,比如应用在蛋白纯化领域,蛋白展示系统等。但是原始的分子肽SpyTag/SpyCatcher的反应条件广泛,不具备对外界环境的刺激响应行为。蛋白质表面的电荷密度的改变可以改变酶的许多特性,其中包括聚集抗性,细胞通透性,刺激响应行为,以及与带相反电荷的大分子结合能力。但是,氨基酸突变对于蛋白质来说都是有风险的,在很大一部分情况下经过改造会出现失活的现象,因此对SpyCatcher的改造也有可能导致无法正常与SpyTag形成异肽键,或影响连接效率。
发明内容
本发明的目的在于提供一种分子肽突变体,在不影响异肽键形成的基础上得到可以对pH有刺激响应的分子肽SpyCatcher-21,并且通过对晶体结构的分析在SpyCatcher-21的关键loop中引入Pro来降低loop的柔性,获得突变体SpyCatcher-21_A82P,在不影响其表面电位的基础上,提高SpyCatcher-21和SpyTag的连接效率。
为实现上述技术目的,本发明采用如下技术方案:
一种分子肽突变体,其特征在于,氨基酸序列如SEQ ID NO:1所示。
本发明的另一目的在于保护编码权利要求1所述分子肽的基因序列。
本发明的又一目的在于提供上述分子肽的在双酶或三酶催化体系中的应用。
具体的,可通过改变环境的pH使SpyCatcher-21_A82P和SpyTag不同程度的偶联用于双酶催化;或是与带有正电的酶通过静电作用相互作用得到三酶偶联的催化体系。
本发明的分子肽可通过如下方式纯化,包括:
(1)将分子肽的基因序列导入载体构建重组质粒,重组质粒导入宿主菌;
(2)将含重组质粒的宿主菌培养至OD600=0.6-0.8,之后加入IPTG诱导;
(3)诱导结束后,取菌液离心后收集菌体,加入磷酸缓冲液重悬,超声破碎;
(4)破碎液超速离心后取上清液,纯化透析获取纯化蛋白。
进一步的,所述(1)中,载体选用pET-22b;载体连接的酶切位点为Nde I和Xho I。
进一步的,所述(1)中,宿主菌为大肠杆菌E. coli BL21(DE3)。
进一步的,所述(2)中,含重组质粒的大肠杆菌在LB培养基中培养。
进一步的,所述(4)中,上清液在Ni-NTA树脂中进行蛋白纯化。
进一步的,纯化后的蛋白在3000 Da的透析袋中透析24-26 h。
本发明的突变体改造原理如下:通过分析SpyTag/SpyCatcher的晶体结构(PDB ID4MLI)发现,SpyTag上的Tyr119和Lys120和SpyCatcher的Tyr84和Glu85分别产生π-π堆积作用和盐桥,对异肽键的形成有着重要的影响。所以我们在SpyCatcher-21的E81-A91 loop上引入一个脯氨酸Pro,由于Pro侧链的刚性较大,会降低E81-A91 loop的柔性,来稳固SpyCatcher-21上Tyr86,Glu87和SpyTagTyr119和Lys120的相互作用,最后在表观呈现出来的是SpyCatcher-21和SpyTag的连接效率提高。
本发明充分考虑氨基酸之间的相互作用力来对SpyCatcher进行设计改造,在尽量不改变核心结构下得到突变体,其连接效率不会受到太大的影响。在原始分子SpyCatcher的基础上,将SpyCatcher进行负电荷改造,在SpyCatcher表面引入了10个酸性氨基酸突变,在不影响异肽键形成的基础上得到带有对pH刺激响应的分子肽SpyCatcher-21,并在SpyCatcher-21的基础上E81-A91 loop上引入一个脯氨酸Pro,获得突变体SpyCatcher-21_A82P,进一步提高了连接效率。本发明的设计的分子肽突变体SpyCatcher-21_A82P可用于在根据客观需要的前提下,通过改变环境的pH来得到不同程度的偶联来得到双酶催化;亦或是可以与带有正电的酶通过静电作用相互作用得到三酶偶联的催化体系。
附图说明
图1是SpyCatcher-21和SpyCatcher-21_A82P序列比对。
图2是SpyCatcher-21_A82P电荷密度图。
图3是SpyCatcher-21电荷密度图。
图4是在不同pH环境下的连接效率
图5是SpyCatcher-21_A82P动力学。
图6是SpyCatcher-21动力学。
具体实施方式
实施例1
本实施例具体说明SpyCatcher-21_A82P的设计方法。
实施例使用的原始SpyCatcher蛋白晶体结构从PDB数据库中获得,PDB ID为4mli。
将SpyCatcher的结构导入计算软件Rosetta中,对SpyCatcher进行计算,将表面电位设置为-21,并且所设置的突变氨基酸位置均在蛋白表面,得到SpyCatcher-21。之后将SpyCatcher-21的82位残基引入Pro得到SpyCatcher-21_A82P突变体。如图1所示。
利用APBS和VMD对蛋白表面的电荷密度进行计算,如图2、图3所示,可以看出,改造获得的分子肽SpyCatcher-21表面带上了大量电荷,突变体SpyCatcher-21_A82P表面同样具备大量电荷,电位和SpyCatcher-21相同。
实施例2
本实施例具体说明SpyCatcher-21和SpyCatcher-21_A82P突变体的纯化方法。
(1)将突变后的分子肽在生工生物工程(上海)股份有限公司进行全基因合成,克隆在载体pET-22b上得到重组质粒SC-21_A82P-pET-22b,酶切位点为Nde I和Xho I,宿主为大肠杆菌E. coli BL21(DE3)。
(2)将带有重组质粒的E. coli BL21(DE3)在LB培养基中37 ℃中培养至OD600=0.6,加入1 M的IPTG至终浓度0.5 mM,20 ℃诱导10 h。
(3)诱导结束后,8000 rpm离心菌液收集菌体,并加入3 mL 磷酸缓冲液,震荡重悬,在300 W的功率下超声破碎10 min。
(4)将破碎液在12000 rpm,4 ℃的环境中超速离心10 min,取上清液,在Ni-NTA树脂中进行蛋白纯化,纯化后的蛋白在3000 Da的透析袋中透析24 h后备用。
实施例3
本实施例测试了不同pH下SpyCatcher-21的连接效率。
按照终浓度10 μM的SpyCatcher-21和30 μM的SpyTag-GFP混合,分别加入pH=4,5,6,7,8,9的缓冲液(0.1 M)在25 ℃环境中反应180 min后,利用SDS-PAGE测得连接效率。
其中蛋白纯化的方法:
将1 mL的Ni-NTA预装柱中20%的乙醇保护液流尽,并且加入3-4倍柱体积的BufferA来替换填料中的乙醇。将超速离心后的蛋白样品倒入中填料中流尽。再加入3-4 柱体积的BufferA进行洗脱,去除吸附在填料上的杂蛋白。再加入3-4倍柱体积的BufferB,洗脱目的蛋白。
BufferA为pH8.0 0.1M的磷酸缓冲液,溶解有500 mM的NaCl和20 mM的咪唑;
BufferB为pH8.0 0.1M的磷酸缓冲液,溶解有500 mM的NaCl和300 mM的咪唑;
1 mL Ni-NTA预装柱购自生工生物工程(上海)股份有限公司。其余试剂均为市售。
SDS-PAGE蛋白凝胶电泳方法:
将30 μL的样品和10 μL的4×loading buffer混合,在100 ℃的金属浴中保温10min,保温结束后降温至4 ℃后1000-12000 rpm离心。利用SDS-PAGE蛋白凝胶试剂盒配置12%的分离胶和5%的浓缩胶。将制备的样品取10 μL上样,电压为120 V,电泳时间为120min。结束后使用考马斯亮蓝染色液染色60-120 min后,利用脱色液脱色至背景透明。在凝胶成像仪拍照,利用ImageJ进行条带密度分析,即可得到连接效率。其中SDS-PAGE蛋白凝胶试剂盒购自北京索莱宝科技有限公司,其余试剂均为市售。
结果如图4所示,SpyCatcher-21具备与SpyTag形成异肽键的能力,在表面带有大量的负电荷,还可以对pH产生刺激响应,SpyCatcher-21_A82P同样具备pH响应。
实施例4
本实施例对比了pH=6时SpyCatcher-21和SpyCatcher-21_A82P的连接效率。
按照终浓度10 μM的SpyCatcher-21_A82P和30 μM的SpyTag-GFP混合,在25 ℃环境中测0,10,20,30,40,50,60,120,180,240 min的SpyCatcher-21_A82P和SpyTag-GFP的连接效率,利用SDS-PAGE测得连接效率。
结果表明,SpyCatcher-21 10 min的连接速率~42%,240 min的连接速率~90%。而通过改造后得到的SpyCatcher-21_A82P可以在10 min达到~80%的连接效率,180 min即可达到100%,SpyCatcher-21_A82P在维持电位、pH响应的基础上大幅提高了连接效率。
序列表
<110> 南京工业大学
<120> 一种分子肽突变体
<130> xb21051202
<141> 2021-05-12
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 115
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ala Met Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser
1 5 10 15
Asp Asp Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile Glu Phe Ser
20 25 30
Lys Arg Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu
35 40 45
Arg Asp Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Asp
50 55 60
Val Lys Asp Phe Tyr Leu Tyr Pro Gly Glu Tyr Thr Phe Val Glu Thr
65 70 75 80
Glu Pro Pro Asp Gly Tyr Glu Val Asp Asp Ala Ile Thr Phe Thr Val
85 90 95
Asn Glu Asp Gly Gln Val Thr Glu Glu Gly Lys Ala Thr Lys Gly Asp
100 105 110
Ala His Ile
115
Claims (10)
1.一种分子肽突变体,其特征在于,氨基酸序列如SEQ ID NO:1所示。
2.编码权利要求1所述分子肽突变体的基因序列。
3.权利要求1所述分子肽突变体在双酶或三酶催化体系中的应用。
4.权利要求1所述分子肽突变体的纯化方法,其特征在于,包括:
(1)将分子肽的基因序列导入载体构建重组质粒,重组质粒导入宿主菌;
(2)将含重组质粒的宿主菌培养至OD600=0.6-0.8,之后加入IPTG诱导;
(3)诱导结束后,取菌液离心后收集菌体,加入磷酸缓冲液重悬,超声破碎;
(4)破碎液超速离心后取上清液,纯化透析获取纯化蛋白。
5.根据权利要求4所述的方法,其特征在于,所述(1)中,载体选用pET-22b。
6. 根据权利要求4所述的方法,其特征在于,载体连接的酶切位点为Nde I和Xho I。
7. 根据权利要求4所述的方法,其特征在于,所述(1)中,宿主菌为大肠杆菌E. coliBL21(DE3)。
8.根据权利要求4所述的方法,其特征在于,所述(2)中,含重组质粒的大肠杆菌在LB培养基中培养。
9.根据权利要求4所述的方法,其特征在于,所述(4)中,上清液在Ni-NTA树脂中进行蛋白纯化。
10. 根据权利要求4所述的方法,其特征在于,纯化后的蛋白在3000 Da的透析袋中透析24-26 h。
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