CN112342248A - 一种基因敲除改变体外蛋白合成能力的方法及其应用 - Google Patents
一种基因敲除改变体外蛋白合成能力的方法及其应用 Download PDFInfo
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Abstract
本发明提供一种真核生物的基因改造方法,其在真核生物的基因组中通过基因编辑技术将Maf1基因敲除。经试验验证,Maf1基因敲除能显著提高无细胞体系的蛋白表达能力。同时,本发明提供包含利用该基因改造方法进行改造的基因工程细胞,以及包含该基因工程细胞制备的细胞提取物的真核无细胞蛋白质合成体系。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种基因敲除可以增强真核生物无细胞蛋白反应体系的蛋白合成能力。
背景技术
合成生物学的概念于上世纪80年代被提出,经过过去几十年的发展,取得了很大的发展。合成生物学中很重要的一个分支是蛋白质的合成,蛋白合成进一步细分为培养的细胞合成体系和无细胞的合成体系。无细胞的蛋白合成体系,是利用蛋白合成所需要的各种酶、蛋白组件和原料,以外源的DNA为模板来合成mRNA和蛋白质的系统。与传统的细胞表达系统相比,无细胞的蛋白合成体系可以表达具有细胞毒性的蛋白或者含有非天然氨基酸的特殊蛋白质;另外其操作更简便,可以进行高通量药物筛选和蛋白质组学研究。鉴于以上所述的诸多优点,无细胞的蛋白合成体系开发越来越多,其应用也被更大范围的拓展。目前商业化的无细胞蛋白合成体系包括原核的大肠杆菌体系,真核的麦胚提取物和兔网织红细胞裂解物体系,但是还没有成熟的酵母细胞提取物体系。由于酵母细胞属于真核细胞,同时酵母细胞可以通过发酵途径大量获取,因此酵母细胞抽提物体系兼具真核系统的蛋白修饰功能和大规模工业化应用的优势,开发基于酵母细胞的无细胞表达系统是极具应用前景的。D2P (DNA to protein)体系是利用乳酸克鲁维的提取物为基础,进行一系列优化得到的无细胞蛋白反应体系,可以填补这方面的空白。
在开发克鲁维酵母抽提物的无细胞蛋白反应体系过程中,一个重要的目标是提高该体系的蛋白质合成产量。影响无细胞系统中蛋白合成的因素很多,比如外源DNA模板的转录强度、转录产物mRNA的稳定性和mRNA模板进行翻译起始的能力,除此之外,反应体系中的氨基酸浓度、tRNA的丰度以及能量代谢的供给也会影响D2P体系的蛋白产量。
在真核酵母细胞中,tRNA主要由聚合酶RNAP III进行转录。不同于RNAPII-mRNA的诸多不同转录调控机制,RNAPIII-tRNA的转录调控是以整体方式进行的。RNAP III的转录活性主要由抑制基因Maf1进行调控。在适合细胞生长的条件下(比如营养充分),Maf1会被PKA,Sch9,TORC1和CK2等激酶进行磷酸化,磷酸化的Maf1大部分定位在细胞质中,从而不会抑制细胞核内的RNAP III的转录功能。在不适合细胞生长的条件下(比如营养不足或温度变化等),Maf1会被去磷酸化,去磷酸化的Maf1在核定位信号的引导下进入细胞核,与RNAPIII结合从而抑制RNAP III的转录活性。
在酿酒酵母细胞的培养发酵过程中,随着细胞密度(OD600)的增加,在生长从指数期进入平台期后,tRNA的丰度会明显下降。由于D2P体系是利用平台期早期的细胞制作的体系,体系中的tRNA丰度可能会比较低,因此通过敲除Maf1基因,解除其对RNAP III的抑制,可以提高D2P体系中的tRNA丰度,从而提高D2P体系的蛋白合成能力。CRISPR-Cas9技术作为高效基因组DNA编辑的工具广泛应用于各种细胞。利用CRISPR-Cas9技术对克鲁维酵母进行Maf1敲除的基因工程改造,并检测Maf1基因敲除的细胞株的蛋白合成能力。
参考文献
1.Magdalena Boguta. Maf1, a general negative regulator of RNA polymeraseIII in yeast. Biochimica Biophysica Acta, 2013, 1829:376–384.
2.Rajendra Upadhya, JaeHoon Lee, Ian M. Willis. Maf1 is an essentialmediator of diverse signals that repress RNA Polymerase III transcription.Molecular Cell, 2002, 10:1489–1494.
3.Gajda A, Towpik J, Steuerwald U, Müller CW, Lefebvre O, Boguta M. Fullrepression of RNA polymerase III transcription requires interaction betweentwo domains of its negative regulator Maf1. J Biol Chem, 2010, 285(46):35719-35727.
4.Moir RD, Lee J, Haeusler RA, Desai N, Engelke DR, Willis IM. Proteinkinase A regulates RNA polymerase III transcription through the nuclearlocalization of Maf1. Proc Natl Acad Sci U S A. 2006, 103(41):15044-15049.
5.Roberts DN, Wilson B, Huff JT, Stewart AJ, Cairns BR. Dephosphorylationand genome-wide association of Maf1 with Pol III-transcribed genes duringrepression. Mol Cell. 2006, 22(5):633-644.
6.Graczyk D, Cieśla M, Boguta M. Regulation of tRNA synthesis by thegeneral transcription factors of RNA polymerase III - TFIIIB and TFIIIC, andby the MAF1 protein. Biochim Biophys Acta Gene Regul Mech. 2018, 1861(4):320-329.
7.Boguta M, Graczyk D. RNA polymerase III under control: repression andde-repression. Trends Biochem Sci. 2011, 36(9):451-456.
8.Vannini A, Ringel R, Kusser AG, Berninghausen O, Kassavetis GA, CramerP. Molecular basis of RNA polymerase III transcription repression by Maf1.Cell. 2010, 143(1):59-70.
9.Willis IM, Moir RD. Integration of nutritional and stress signalingpathways by Maf1. Trends Biochem Sci. 2007, 32(2):51-53。
发明内容
鉴于此,本发明提供一种提高体外无细胞蛋白合成体系的蛋白合成能力的方法,从分子水平提高无细胞蛋白合成体系的蛋白合成能力,从而能够进一步实现无细胞蛋白合成体系节约成本、操作简单的目的。
本发明主要包括以下几个方面:
第一方面,提供一种改变体外无细胞蛋白合成体系的蛋白合成能力的方法,所述方法包括以下步骤:
(1)将待改造的真核细胞中原始存在的Maf1基因通过基因编辑技术去除,获得ΔMaf1改造菌株;
(2)以上述ΔMaf1改造菌株制备细胞裂解液或细胞提取物;
(3)将步骤(2)获得的细胞裂解液或细胞提取物用于体外无细胞蛋白质合成体系。
进一步的,所述真核细胞为哺乳动物细胞、植物细胞、酵母细胞、昆虫细胞之一或其任意组合。
进一步的,所述酵母细胞选自酿酒酵母、毕氏酵母、克鲁维酵母之一或其任意组合。
进一步的,所述克鲁维酵母属酵母选自乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母之一或其任意组合。
第二方面,提供一种体外无细胞蛋白合成体系,所述合成体系至少包括以下组分:细胞裂解液或细胞提取物,所述细胞裂解液或细胞提取物由ΔMaf1改造菌株制备获得,所述ΔMaf1改造菌株为将待改造的真核细胞中原始存在的Maf1基因通过基因编辑技术去除获得。
进一步的,第二方面的所述合成体系还包括选自下组的一种或多种组分:用于合成RNA的底物,用于合成蛋白的底物,聚乙二醇或其类似物,镁离子,钾离子,缓冲剂,RNA聚合酶,能量再生系统,二硫苏糖醇(DTT),任选的水或水性溶剂。
第三方面,提供一种合成外源蛋白的方法,所述方法包括以下步骤:
(i) 提供第二方面所述的体外无细胞蛋白合成体系;
(ii) 添加编码外源蛋白的DNA分子模板,在适合的条件下,孵育一段时间,合成所述的外源蛋白。
进一步的,第三方面的所述方法还包括:(iii)分离或检测所述外源蛋白。
进一步的,合适的条件包括反应温度为20-35℃,优选的为20-30℃,更优选的为25℃。
进一步的,孵育一段时间具体为0.5-20h,优选的为1-18h,更优选的为2-15 h,更优选的为3-12h;上述反应时间可根据具体情况人为确定,也可以为3-15h,也可以为3-20h,也可以为具体的时间点,如3h、5h、10h、15h、18h、20h。
第四方面,提供一种试剂盒,所述的试剂盒包括容器以及位于所述容器中的第二方面所述的体外无细胞蛋白合成体系中的组分。
本发明的主要优点包括:
(1)本发明通过基因定向改造以及活性测定,首次验证敲除Maf1基因可以提高体外无细胞蛋白合成体系的蛋白合成能力;
(2)本发明通过CRISPR-Cas9基因编辑技术对Maf1进行基因敲除改造,从而改变体外蛋白质合成能力。
附图说明
图1是pCas9_KlMaf1_gRNA1图谱示意图。该质粒带有K.lactis SNR52启动子和SNR52终止子,并带有Kana筛选标记。
图2是pCas9_KlMaf1_gRNA2图谱示意图。
图3是pMD18T-ΔKlMaf1的质粒图谱示意图。KlMaf1的起始密码子上游899bp是HR1,终止密码子下游的873bp是HR2,质粒带有Amp筛选标记。
图4是ΔKlMaf1菌株和野生型菌株D2P反应体系绿色荧光蛋白合成量的比较。
具体实施方式
本发明人经过广泛而深入的研究,发现Maf1基因敲除的细胞与野生型未改造细胞相比,能显著提高体外无细胞蛋白合成体系的蛋白合成能力,提高外源蛋白的表达产量。
术语
为了可以更容易地理解本公开,首先定义某些术语。如本申请中所使用的,除非本文另有明确规定,否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成,其将部分地取决于如何测量或测定值或组成。例如,如本文所用,表述“约100”包括99和101和之间的全部值(例如,99.1、99.2、99.3、99.4等)。
体外无细胞蛋白合成体系(D2P)
本发明提供了一种表达外源蛋白的体外无细胞蛋白合成体系,所述合成体系主要至少包括:细胞裂解液或细胞提取物;所述细胞裂解液或细胞提取物来自于所述的Maf1敲除工程细胞,该工程细胞提取物中不含有Maf1基因的表达产物,即为ΔMaf1改造菌株制备细胞裂解液或细胞提取物。
进一步的,所述合成体系还包括选自下组的一种或多种组分:用于合成蛋白质的底物、用于合成RNA的底物、RNA聚合酶、镁离子、钾离子、缓冲剂、能量再生系统、聚乙二醇(PEG)或其类似物、二硫苏糖醇(DTT)和任选的溶剂,所述溶剂为水或水性溶剂。
进一步的,所述细胞为真核细胞。所述真核细胞为哺乳动物细胞、植物细胞、酵母细胞、昆虫细胞之一或其任意组合。其中,所述酵母细胞选自酿酒酵母、毕氏酵母、克鲁维酵母之一或其组合;克鲁维酵母属酵母选自乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母之一或其任意组合;较佳地,所述的酵母细胞为克鲁维酵母,更佳地为乳酸克鲁维酵母。
进一步的,所述细胞提取物为对酵母细胞的水性提取物。
进一步的,所述细胞提取物不含酵母内源性的长链核酸分子。
进一步的,所述的合成RNA的底物包括:核苷单磷酸、核苷三磷酸之一或其组合。
进一步的,所述的合成蛋白质的底物包括:20种天然氨基酸以及非天然氨基酸。
进一步的,所述镁离子来源于镁离子源,所述镁离子源选自下组:醋酸镁、谷氨酸镁之一或其组合。
进一步的,所述钾离子来源于钾离子源,所述钾离子源选自下组:醋酸钾、谷氨酸钾之一或其组合。
进一步的,所述能量再生系统选自下组:磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统之一、蔗糖或其组合。
进一步的,所述缓冲剂选自下组:4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷之一或其组合。
进一步的,所述蛋白合成体系含有聚乙二醇(PEG)或其类似物。聚乙二醇或其类似物的浓度没有特别限制,通常,聚乙二醇或其类似物的浓度 (w/v)为0.1-8%,较佳地,0.5-4%,更佳地,1-2%,以所述蛋白合成体系的总重量计。代表性的PEG选自下组:PEG3000、PEG3350、PEG6000、PEG8000之一或其组合。
进一步的,所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇,如PEG200、400、1500、2000、4000、6000、8000、10000等,较佳地,分子量为3000-10000的聚乙二醇。
可选的方案为,本发明提供的蛋白合成体系包括:酵母细胞提取物,4-羟乙基哌嗪乙磺酸,醋酸钾,醋酸镁,腺嘌呤核苷三磷酸(ATP),鸟嘌呤核苷三磷酸(GTP),胞嘧啶核苷三磷酸(CTP),胸腺嘧啶核苷三磷酸(TTP),氨基酸混合物,磷酸肌酸,二硫苏糖醇(DTT),磷酸肌酸激酶,RNA聚合酶,聚乙二醇,蔗糖。
在本发明中,所述的细胞提取物不含完整的细胞,典型的细胞提取物包括用于蛋白翻译的核糖体、转运RNA、氨酰tRNA合成酶、蛋白质合成需要的起始因子和延伸因子以及终止释放因子。此外,细胞提取物中还含有一些源自细胞的细胞质中的其他蛋白,尤其是可溶性蛋白。
在本发明中,所述的细胞提取物所含蛋白含量为20-100mg/ml, 较佳为50-100mg/ml。所述的测定蛋白含量方法为考马斯亮蓝测定方法。
在本发明中,所述的细胞提取物或细胞裂解液的制备方法不受限制,一种优选的制备方法包括以下步骤:
(i)提供细胞;
(ii)对细胞进行洗涤处理,获得经洗涤的细胞;
(iii)对经洗涤的细胞进行细胞破碎处理,从而获得细胞粗提物;
(iv)对所述细胞粗提物进行固液分离,获得液体部分,即为细胞提取物。
在本发明中,所述的固液分离方式不受特别限制,一种优选的方式为离心。
在本发明中,所述离心条件不受特别限制,一种优选的离心条件为5000-100000×g,较佳地,8000-30000×g。
在本发明中,所述离心时间不受特别限制,一种优选的离心时间为0.5min-2h,较佳地,20min-50min。
在本发明中,所述离心的温度不受特别限制,优选的,所述离心在1-10℃下进行,较佳地,在2-6℃下进行。
在本发明中,所述的洗涤处理方式不受特别限制,一种优选的洗涤处理方式为采用洗涤液在pH为7-8(较佳地,7.4)下进行处理,所述洗涤液没有特别限制,典型的所述洗涤液选自下组:4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在本发明中,所述细胞破碎处理的方式不受特别限制,一种优选的所述的细胞破碎处理包括高压破碎、冻融(如液氮低温)破碎。
所述蛋白合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中,各种单核苷酸的浓度没有特别限制,通常每种单核苷酸的浓度为0.5-5mM,较佳地为1.0-2.0mM。
所述蛋白合成体系中的氨基酸混合物可包括天然或非天然氨基酸,可包括D型或L型氨基酸。代表性的氨基酸包括(但并不限于) 20种天然氨基酸:甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。每种氨基酸的浓度通常为0.01-0.5mM,较佳地0.02-0.2mM,如0.05、0.06、0.07、0.08 mM。
在优选例中,所述体外无细胞蛋白合成体系还含有蔗糖,所述蔗糖的浓度为0.03-40wt%,较佳地,0.08-10wt%,更佳地,0.1-5wt%,以所述蛋白合成体系的总重量计。
一种特别优选的体外无细胞蛋白合成体系,除了酵母细胞提取物之外,还含有以下组分:22 mM pH为7.4的4-羟乙基哌嗪乙磺酸,30-150 mM醋酸钾,1.0-5.0 mM醋酸镁,1.5-4 mM核苷三磷酸混合物,0.08-0.24 mM的氨基酸混合物,25 mM磷酸肌酸,1.7mM二硫苏糖醇,0.27mg/mL磷酸肌酸激酶, 1%-4%聚乙二醇,0.5%-2%蔗糖, 0.027-0.054 mg/mL T7RNA聚合酶。
细胞提取物
本发明中细胞提取物和细胞裂解液所表达的含义相同,均为细胞破碎以后所获得的物质。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人, 分子克隆:实验室手册(New York: Cold Spring HarborLaboratory Press, 1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。
除非有特别说明,否则本发明实施例中的试剂和材料均为市售产品。
本发明以乳酸克鲁维酵母(Kluyveromyces lactis,K. lactis)为实施例,但同样的设计和分析、实验方法也适用于其他酵母等低等真核细胞以及高等动物细胞。本发明中的基因改造方法为CRISPR-Cas9技术,但并不限于此,可以为已知的、现有的任意基因改造方法。
实施例1 通过CRISPR-Cas9敲除酵母Maf1基因
1.1 Maf1序列检索及CRISPR gRNA质粒的构建
根据S.cerevisiae的Maf1蛋白序列比对,确定K.lactis细胞中Maf1的基因编号为KLLA0_E17535g(uniprot数据库,https://www.uniprot.org/),其核苷酸序列为SEQ No.1,氨基酸序列为SEQ No.2。在Maf1基因编码序列靠近5’和3’分别选择PAM序列(NGG),并确定对应的gRNA序列。本实施例中gRNA选择的原则为:GC含量适中(40%-60%),避免poly T结构的存在。在本实施例中,KlMaf1 gRNA1序列为CTGTTAGCGAGAATTCGAGT,KlMaf1 gRNA2序列为AGATTTCAAGGATGAAGCCA。
KlMaf1 gRNA1的质粒构建及转化方法如下:使用引物pCas9-KlMaf1-gRNA1-PF:CTGTTAGCGAGAATTCGAGTGTTTTAGAGCTAGAAATAGC和pCas9-KlMaf1-gRNA1-PR:ACTCGAATTCTCGCTAACAGAAAGTCCCATTCGCCACCCG,以pCAS质粒为模板,进行PCR扩增。取扩增产物17 µL,加入0.2 µL Dpn I,2 µL 10 × digestion buffer,混匀后37℃水浴3 h。取Dpn I处理后产物10 µL加入50 µL DH5α感受态细胞中,冰上放置30 min,42℃热激45 s后,加入1 mL LB液体培养基37℃振荡培养1 h,涂布于Kan抗性LB固体培养,37℃倒置培养至单克隆长出。挑取2个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pCas9_KlMaf1_gRNA1。
KlMaf1 gRNA2的构建方法如下:使用引物pCas9-KlMaf1-gRNA2-PF: AGATTTCAAGGATGAAGCCAGTTTTAGAGCTAGAAATAGC和pCas9-KlMaf1-gRNA2-PR:TGGCTTCATCCTTGAAATCTAAAGTCCCATTCGCCACCCG,以pCAS质粒为模板,进行PCR扩增。转化及鉴定方法同上,阳性质粒命名为pCas9_KlMaf1_gRNA2。
1.2 Maf1敲除供体DNA质粒构建及扩增
为了便于线性供体DNA的保存及扩增,本实施例将供体DNA插入到pMD18T质粒中,并通过PCR扩增得到线性供体DNA序列。
以pMD18T质粒为模板,以引物pMD18T-PF:ATCGTCGACCTGCAGGCATG和pMD18T-PR:ATCTCTAGAGGATCCCCGGG进行PCR扩增,取扩增产物17 µL,加入1 µL DpnI,2 µL 10 ×digestion buffer,混匀后37℃水浴3 h,获得质粒骨架线性片段pMD18T-vector。
1.2.1 构建供体质粒pMD18T-ΔKlMaf1
以乳酸克鲁维酵母基因组DNA为模板,用引物KlMaf1-HR1-PF:CAGGAAACAGCTATGACTACCCGGGGATCCTCTAGAGATCACTCACAGAGCAAGCTCCTCTC和KlMaf1-HR1-PR:CCTTCTCTTTTGTTCCCAGAATG进行PCR扩增,产物名为Maf1-HR1;以乳酸克鲁维酵母基因组DNA为模板,利用引物KlMaf1-HR2-PF:CATTCTGGGAACAAAAGAGAAGGTTATCATTACATTTTCGAACC和KlMaf1-HR2-PR:GTAAAACGACGGCCAGTTGCATGCCTGCAGGTCGACGATGATGAGACCATATTGAGTGACG进行PCR扩增,产物名为Maf1-HR2;
将扩增产物Maf1-HR1、Maf1-HR2和pMD18T-vector各1µL,加入3µL Cloning Mix(Transgene pEASY-Uni Seamless Cloning and Assembly Kit,全式金公司,下同),混匀后50℃水浴1h。水浴结束后置于冰上2min,将6µL反应液全部加入50µL Trans-T1感受态细胞(全式金公司,下同)中,冰上放置30min,42 ℃热激30 s后,加入1 mL LB液体培养基37℃振荡培养1h,涂布于Amp抗性LB固体培养,37℃倒置培养至单克隆长出。挑取6个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pMD18T-ΔKlMaf1。
1.3 K. lactis电转化
从-80℃冰箱取出感受态,冰上融化,加入200 ng质粒pCas9_KlMaf1_gRNA1,200ng质粒pCas9_KlMaf1_gRNA2和通过引物对pMD18T-ΔKlMaf1扩增得到的1000 ng 线性供体DNA片段,混匀后全部转入电击杯中,冰浴 2 min;将电击杯放入电转仪中进行电击(参数为1.5kV,200 Ω,25μF);电击结束后立即加入 700μL YPD,30℃,200rpm摇床孵育1~3 h;取2~200 μL接种于YPD(含G418抗性)平板,30℃培养2~3天至单菌落出现。
1.4 阳性鉴定
在细胞转化后的平板上挑取12-24个单克隆,以细胞基因组为模板,用鉴定引物ΔMaf1-CF: TCGCAAAGGATCAAGGAGCAG和ΔMaf1-CR: TGGGCTGGAACATCGGATGG对样品进行PCR检测。阳性菌株条带1916bp,阴性菌株条带2906bp。经测序鉴定的阳性细胞株,命名为ΔKlMaf1。
实施例2体外蛋白质合成体系
2.1 细胞提取物的制备
细胞提取液的制备方法包括以下步骤:
(i)提供细胞,细胞为实施例1制备的ΔKlMaf1细胞株;
(ii)对细胞进行洗涤处理,获得经洗涤的细胞;
(iii)对经洗涤的细胞进行细胞破碎处理,从而获得细胞粗提物;
(iv)对所述细胞粗提物进行固液分离,获得液体部分,即为细胞提取物。
其中,固液分离方式不受特别限制,本实施例选择的方式为离心。离心条件为30000×g;离心时间为30min;离心4oC下进行。
其中,洗涤处理方式不受特别限制,本实施例选择的洗涤处理方式为采用洗涤液在pH为7.4下进行处理,所述洗涤液没有特别限制,典型的所述洗涤液选自下组:4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。本实施例选择醋酸钾。
其中,细胞破碎处理的方式不受特别限制,一种优选的所述的细胞破碎处理包括高压破碎、冻融(如液氮低温)破碎。
体外蛋白质合成体系的配制
终浓度为22 mM pH为7.4的4-羟乙基哌嗪乙磺酸,30-150 mM醋酸钾,1.0-5.0 mM醋酸镁,1.5-4 mM核苷三磷酸混合物(腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸),0.08-0.24 mM的氨基酸混合物(甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸),25 mM磷酸肌酸,1.7 mM二硫苏糖醇,0.027-0.054 mg/mL T7 RNA 聚合酶,0.27 mg/mL磷酸肌酸激酶, 1% - 4% 的聚乙二醇,0.5% - 2% 的蔗糖,最后加入50 %体积的细胞提取物。
体外蛋白质合成反应
将上述的反应体系放置在约为25℃的环境中,反应20h;
增强型绿色荧光蛋白(eGFP)活性测定:反应结束后,在96孔白板或384孔白板中加入10µL反应液,立即放置于Envision 2120多功能酶标仪(Perkin Elmer), 读数,检测增强型绿色荧光蛋白活性,相对荧光单位值(Relative Fluorescence Unit,RFU)作为活性单位,如图4所示。
其中ΔKlMaf1表示Maf1敲除的细胞株,WT表示野生型的乳酸克鲁维酵母细胞株。反应时间为20h时,ΔKlMaf1的RFU平均值为3708,WT的RFU平均值是2980,ΔKlMaf1是WT的约1.244倍。该对比数据表明通过基因编辑技术敲除Maf1基因,能够显著提高的蛋白合成能力。
本发明实施例结果表明:
与野生型细胞株相比,本发明的Maf1基因敲除细胞株体外无细胞蛋白合成反应体系的蛋白合成能力提高了约24.4%。
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 康码(上海)生物科技有限公司
<120> 一种基因敲除改变体外蛋白合成能力的方法及其应用
<130> 2019
<141> 2019-08-08
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 990
<212> DNA
<213> 乳酸克鲁维酵母(Kluyveromyces lactis)
<400> 1
atgtctgtta gcgagaattc gagtgggaca aatagttcgt cacctataat aaaatctaat 60
aagttgaacg accaaaattt gaaggaattc gtgctgtcaa ctgacagtac aaattttgtc 120
acgaatggac acaacaatag taatagtagt accaccaatg ctaactccaa taacggatat 180
ttgagttcat catcaatgga atcggggcct ttagggatcg cttcacaacg tcccagtttc 240
ctaaggagga ctagcagtgc tggaagcatt gcaagcaacc ataccgatac ttcgaaaaga 300
agacctagca caagtggttc agcgaatgaa atacaaaata cttcatacca acagaggagg 360
aggtcatcgg tacaaggaag agacaacgta aatattggac cgtttggacc tatcaacgaa 420
actgctagca ggagagcttt cgcgtatcta atagccattt tgaatgcgtc ctatccggat 480
cacgattttt cgtcattaga gcccaccgat ttcgtgaaat gcaccaagaa aagtttcatc 540
tcgaagttcg aaaatacctt gatatcttta ggaaaaccac ctcaggaatg gatctgggaa 600
acgatcaacg gacatatgga tatcaacgat tgcgctttct atcaatatgt ccctcaaaaa 660
tcgtttcttg aagatgagcc tggccatctt tggtcattga tgtggttttt gttcaataaa 720
aaaaggaaaa gagtcgcata tgtttatcta acagcatgcc gtttgaaagg aactccctct 780
gttggaaata gtgtcatatt ggcggatgac gaagatgaaa attcaagtaa actagaatca 840
gaacgtaaac agagaaggaa attgacgtta gataacgaca acgattttga aggcgaatat 900
gatttaactt atgaggagaa tgctataatt gatgatgatg agggcatcaa cgaagatttc 960
aaggatgaag ccatggatga tcttaactga 990
<210> 2
<211> 329
<212> PRT
<213> 乳酸克鲁维酵母(Kluyveromyces lactis)
<400> 2
Met Ser Val Ser Glu Asn Ser Ser Gly Thr Asn Ser Ser Ser Pro Ile
1 5 10 15
Ile Lys Ser Asn Lys Leu Asn Asp Gln Asn Leu Lys Glu Phe Val Leu
20 25 30
Ser Thr Asp Ser Thr Asn Phe Val Thr Asn Gly His Asn Asn Ser Asn
35 40 45
Ser Ser Thr Thr Asn Ala Asn Ser Asn Asn Gly Tyr Leu Ser Ser Ser
50 55 60
Ser Met Glu Ser Gly Pro Leu Gly Ile Ala Ser Gln Arg Pro Ser Phe
65 70 75 80
Leu Arg Arg Thr Ser Ser Ala Gly Ser Ile Ala Ser Asn His Thr Asp
85 90 95
Thr Ser Lys Arg Arg Pro Ser Thr Ser Gly Ser Ala Asn Glu Ile Gln
100 105 110
Asn Thr Ser Tyr Gln Gln Arg Arg Arg Ser Ser Val Gln Gly Arg Asp
115 120 125
Asn Val Asn Ile Gly Pro Phe Gly Pro Ile Asn Glu Thr Ala Ser Arg
130 135 140
Arg Ala Phe Ala Tyr Leu Ile Ala Ile Leu Asn Ala Ser Tyr Pro Asp
145 150 155 160
His Asp Phe Ser Ser Leu Glu Pro Thr Asp Phe Val Lys Cys Thr Lys
165 170 175
Lys Ser Phe Ile Ser Lys Phe Glu Asn Thr Leu Ile Ser Leu Gly Lys
180 185 190
Pro Pro Gln Glu Trp Ile Trp Glu Thr Ile Asn Gly His Met Asp Ile
195 200 205
Asn Asp Cys Ala Phe Tyr Gln Tyr Val Pro Gln Lys Ser Phe Leu Glu
210 215 220
Asp Glu Pro Gly His Leu Trp Ser Leu Met Trp Phe Leu Phe Asn Lys
225 230 235 240
Lys Arg Lys Arg Val Ala Tyr Val Tyr Leu Thr Ala Cys Arg Leu Lys
245 250 255
Gly Thr Pro Ser Val Gly Asn Ser Val Ile Leu Ala Asp Asp Glu Asp
260 265 270
Glu Asn Ser Ser Lys Leu Glu Ser Glu Arg Lys Gln Arg Arg Lys Leu
275 280 285
Thr Leu Asp Asn Asp Asn Asp Phe Glu Gly Glu Tyr Asp Leu Thr Tyr
290 295 300
Glu Glu Asn Ala Ile Ile Asp Asp Asp Glu Gly Ile Asn Glu Asp Phe
305 310 315 320
Lys Asp Glu Ala Met Asp Asp Leu Asn
325
Claims (10)
1.一种改变体外无细胞蛋白合成体系的蛋白合成能力的方法,其特征在于:所述方法包括以下步骤:
(1)将待改造的真核细胞中原始存在的Maf1基因通过基因编辑技术去除,获得ΔMaf1改造菌株;
(2)以上述ΔMaf1改造菌株制备细胞裂解液或细胞提取物;
(3)将步骤(2)获得的细胞裂解液或细胞提取物用于体外无细胞蛋白质合成体系。
2.根据权利要求1所述的方法,其特征在于:所述真核细胞为哺乳动物细胞、植物细胞、酵母细胞、昆虫细胞之一或其任意组合。
3.根据权利要求2所述的方法,其特征在于:所述酵母细胞选自酿酒酵母、毕氏酵母、克鲁维酵母之一或其任意组合。
4.根据权利要求3所述的方法,其特征在于:所述克鲁维酵母属酵母选自乳酸克鲁维酵母、马克斯克鲁维酵母、多布克鲁维酵母之一或其任意组合。
5.一种体外无细胞蛋白合成体系,其特征在于:所述合成体系至少包括以下组分:细胞裂解液或细胞提取物,所述细胞裂解液或细胞提取物由ΔMaf1改造菌株制备获得,所述ΔMaf1改造菌株为将待改造的真核细胞中原始存在的Maf1基因通过基因编辑技术去除获得。
6.根据权利要求5所述的合成体系,其特征在于:所述合成体系还包括选自下组的一种或多种组分:用于合成RNA的底物,用于合成蛋白的底物,聚乙二醇或其类似物,镁离子,钾离子,缓冲剂,RNA聚合酶,能量再生系统,二硫苏糖醇,任选的水或水性溶剂。
7.一种合成外源蛋白的方法,其特征在于,所述方法包括以下步骤:
(i) 提供权利要求5-6任一项所述的体外无细胞蛋白合成体系;
(ii) 添加编码外源蛋白的DNA分子模板,在适合的条件下,孵育一段时间,合成所述的外源蛋白。
8.根据权利要求7所述的方法,其特征在于,合适的条件包括反应温度为20-35℃。
9.根据权利要求7或8任一项所述的方法,其特征在于,所述方法还包括:(iii)分离或检测所述外源蛋白。
10.一种试剂盒,其特征在于,所述的试剂盒包括容器以及位于所述容器中的权利要求5-6任一项所述的体外无细胞蛋白合成体系的组分。
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