CN101638642B - 提高了热稳定性及蛋白酶抗性的环化植酸酶 - Google Patents
提高了热稳定性及蛋白酶抗性的环化植酸酶 Download PDFInfo
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Abstract
本发明涉及一种提高了热稳定性及蛋白酶抗性的环化酶，本发明还涉及对蛋白酶进行环化以提高其性能的方法。本发明利用天然内含肽转剪接环化蛋白质，环化后的蛋白质具有更好的热稳定性和蛋白酶抗性。本发明还提供了含有本发明的植酸酶基因的重组大肠杆菌细胞。
Description
技术领域
本发明涉及一种提高了热稳定性及蛋白酶抗性的环化酶，本发明还涉及对蛋白酶进行环化以提高其性能的方法。
背景技术
天然蛋白质，尤其是酶蛋白在稳定性方面的存在许多不足，比如耐热性差，易被蛋白酶水解等，限制了它们在医药和工业生产中的应用。
例如，植酸酶(E.C.3.1.3.8)是一种可以将植酸水解为肌醇和磷酸的酶类。饲料中添加植酸酶后可将植酸和植酸盐水解成肌醇和磷酸盐，供动物吸收利用，从而提高磷的利用率和骨骼矿化程度，可节约磷资源、降低饲料成本。但植酸酶对热不稳定，而植酸酶生产过程的制粒环节，温度为75-90℃，并要持续数分钟，在此过程中酶的活性会大幅度的丧失，影响其在生产中的应用。因此提高植酸酶的稳定性成为急需解决的问题。
蛋白环化是一种新技术，环化的蛋白较之线型的蛋白有很多的好处，例如可增加蛋白的稳定性，减少蛋白对水解酶剪切的抗性，增强蛋白的活性和减少蛋白构象的柔性等。
应用工程内含肽来环化蛋白质，即应用细菌表达系统生产环化蛋白，这种方法开辟了改进蛋白质稳定性的途径。这种环化方法通过蛋白剪接反应实现的，从蛋白前体上释放一个内部的蛋白序列，称为内含肽。在内部蛋白序列的剪接过程中，内含肽被包埋蛋白前体中，催化自身的剪切，进而把两侧的蛋白序列连接起来。将目的的蛋白融合表达在内含肽的两部分中，形成一个三明治结构。当内含肽被释放时，目的蛋白的C、N端处形成一个肽键，使目的蛋白环化起来，蛋白质的稳定性将会提高。
发明内容
本发明的总的目的是开发一种新的酶蛋白环化技术，提供具有热稳定性及蛋白酶抗性的环化酶。
本发明的另一目的是提供在细菌表达系统中高效表达生产环化酶的方法。
技术解决方案
本发明提供了一种环化的酶，其特征在于，其多肽序列的前5个氨基酸具有SEQ IDNO：1序列，最后5个氨基酸具有SEQ ID NO：2序列；且酶蛋白的N、C两端相链接。
所述环化的酶蛋白的N、C两端相链接，是通过内含肽介导的环化方法，用天然的肽键将酶蛋白的N、C两端链接起来。
在本发明的一个实例中，对枯草芽孢杆菌(Bacillus subtilis)的中性植酸酶基因PhyC进行环化，编码该环化植酸酶的基因具有如SEQ ID NO：3所示的核苷酸序列。经实验证实，环化后的植酸酶的热稳定性和蛋白酶的抗性均有所提高。
本发明还提供了一种环化酶蛋白的方法，包括整套表达载体的构建、重组字的筛选及环化分子的鉴定方法，将前5个氨基酸具有SEQ ID NO：1序列，最后5个氨基酸具有SEQ ID NO：2序列的编码基因在大肠杆菌中的环化表达。
所述环化酶蛋白的方法是利用天然集胞蓝细菌(Synechocystis species PCC6803)Ssp DnaE内含肽的转剪接特性，分别于体内体外对蛋白质环化。
在本发明的一个实例中，所述酶蛋白是植酸酶蛋白，环化方法是将SEQ ID NO：3所示核苷酸序列的基因在大肠杆菌中的环化表达，通过内含肽的转剪接作用环化中性植酸酶PhyC，所述Ssp DnaE内含肽的基因具有如SEQ ID NO：4所示的核苷酸序列。具体方法如下：
1.环化载体构建按常规方法克隆(New York：Cold Spring Harbor LaboratoryPress，1989)。从集胞蓝细菌(Synechocystis species PCC6803)的dnaE基因中PCR，分别得到Ssp DnaE内含肽的N端和C端2部分，后连入pMXB10载体，形成表达载体pEB1。
2.基因克隆从枯草芽孢杆菌(Bacillus subtilis)的基因组中PCR得到一个中性植酸酶基因phyC，克隆到上述表达载体pEB1中。转化到大肠杆菌，获得重组子。
3.蛋白的表达和纯化加入IPTG诱导表达PhyC植酸酶，通过镍柱亲和纯化体内生成的环形植酸酶；通过几丁质亲和柱得到体外的环形植酸酶。验证蛋白的环化情况。
用本发明的方法改良的植酸酶具有更好的热稳定性。本方法为体内环型植酸酶的生产奠定了基础。
附图说明
图1为大肠杆菌重组载体pPEB的物理图谱；
图2为大肠杆菌重组载体pEB1的物理图谱；
图3表示40～75℃下，环化植酸酶cPhyC和野生植酸酶PhyC在各温度保温1小时后，得出的环化植酸酶与野生植酸酶的热稳定性；
图4环化植酸酶的最适反应温度；
图5环化植酸酶对外肽酶的抗性。
具体实施方式
下面结合具体实施例进一步阐述本发明。应理解，该实施例仅用于举例说明本发明的方法，而不限制所用酶蛋白的范围。
实验条件
菌株与载体大肠杆菌菌株E.coli BL21(DE3)购自Novagen公司，载体由本研究构建并保存。
酶与试剂盒限制性内切酶，连接酶，Taq酶为NEB公司产品。基因组提取试剂盒为天根生化公司产品。凝血酶为Merk公司产品。
生化试剂DNA合成试剂为Milipore公司产品。引物合成为ABI公司Cyclone DNA合成仪。IPTG、X-Gal、SDS及植酸酶钠为Sigma公司产品。TEMED、过硫酸铵、丙烯酰胺及甲叉双丙烯酰胺为Promega公司产品。NTA树脂为Qiagen公司产品。几丁质树脂为NEB公司产品。
培养基大肠杆菌培养基为LB(1％蛋白胨、0.5％酵母提取物、1％NaCl，pH7.0)。
实施例中其它未注明具体条件的实验方法，按照常规方法进行，如Sambrook等人的方法，分子克隆按(New York：Cold Spring Harbor Laboratory Press，1989)实验室手册中所述的条件，或按照制造厂商所建议的条件。
实施例1环化植酸酶
1)环化表达载体的构建
用于环化的大肠杆菌表达载体pEB1。首先提取集胞蓝细菌(Synechocystis species)的基因组。以所提取的基因组为模板，PCR扩增dnaE的C端序列。所使用的引物根据dnaE的C段基因的5’和3’端序列合成，2个引物序列分别为：5’ATGGTTAAAGTTATCGGTC 3’和5’GTTAGCTGCGATAGCGCC 3’，扩增后进行凝胶电泳，回收大小108bp的片段。另外，以所提取的基因组为模板，PCR扩增dnaE的N端序列。所使用的引物根据dnaE的N段基因的5’和3’端序列合成，2个引物序列分别为：5’TGCCTGTCTTTTGGTACC 3’和5’TTTAATTGTC CCAGCGTC 3’，扩增后进行凝胶电泳，回收大小369bp的片段。之后将dnaE的C段基因经HindIII和AflII酶酶切，dnaE的N段基因经AflII和EcoRI酶切后连入pMXB10载体，形成pEBl载体(见图2)，dnaE的C段基因前为T7启动子。
2)植酸酶基因分子的克隆
提取枯草芽孢杆菌(Bacillus subtilis)基因组。以所提取的基因组为模板，进行PCR扩增，电泳回收植酸酶phyc基因约1124bp片段。所使用的引物根据phyc基因的5’和3’端序列合成，2个引物序列分别为：
5’CTTAAGCACCATCATCATCATCATAAGCATAAGCTGTCCGATC 3’和
5’ATCGATGCTACCACGCGGAACCAGTTTTCC GCTTCTGTC 3’，
扩增后进行凝胶电泳，回收大小1124bp的片段，SEQ ID NO：3为植酸酶phyC的碱基序列，SEQ ID NO：5为其氨基酸序列。获得的DNA分子经AflII酶切，与经酶切的实验一中得到的pEB1载体于4℃连接过夜，转化大肠杆菌BL21(DE3)，LB培养基涂板后筛选阳性重组子，提取质粒进行鉴定，得到重组子。重组质粒pPEB图谱见图1。
3)环化植酸酶表达纯化的程序
将实验二得到的重组子接种到20mL的LB培养基中，37℃振荡培养过夜，按10％接种量接种于1LB培养基中，与37℃培养至OD为0.6，加入IPTG至终浓度0.5mM，继续培养3小时，诱导表达植酸酶。离心收集菌体，菌体重悬于100mL含500mM NaCl的Tris-HCl(pH7.0)缓冲液中，超声波破碎菌体，15000rpm离心30分钟去除细胞碎片。上清液通过NTA镍株进行纯化。将NTA树脂装入合适的层析柱，层析用10倍NTA体积的NTA-0Buffer(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油)洗。将样品离心后上清加到NTA层析柱中，流速控制在15ml/小时左右，收集穿透部分，用于SDS/PAGE分析蛋白质的结合情况。层析用5倍NTA体积的NTA-0Buffer洗，流速控制在30ml/小时左右。分别用5倍NTA体积的NTA-20(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油，20mM咪唑)，NTA-40(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油，40mM咪唑)，NTA-60(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油，60mM咪唑)，NTA-80(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油，80mM咪唑)，NTA-100(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油，100mM咪唑)缓冲液洗脱，流速控制在15ml/小时左右，收集洗脱液，每管收集一个NTA体积。SDS/PAGE确定目标蛋白质在洗脱液中的分布情况。
4)植酸酶体外环化及纯化
将实验二得到的重组子接种到20mL的LB培养基中，37℃振荡培养过夜，按10％接种量接种于1LB培养基中，与37℃培养至OD为0.6，加入IPTG至终浓度0.5mM，继续培养3小时，诱导表达植酸酶。离心收集菌体，菌体重悬于100mL含500mM NaCl的Tris-HCl(pH7.0)缓冲液中，超声波破碎菌体，15000rpm离心30分钟去除细胞碎片。将上清液慢加入几丁质柱，之后用20倍柱床体积的缓冲液1(20mM Tris-HCl pH7.9，0.5MNaCl)彻底洗柱。停止柱流23℃放置15小时。后用缓冲液1将目的蛋白洗脱收集。大约收集8-10管，每管5ml。通过280nm UV吸光值或Bradford蛋白检测法进行检测。
实施例2环化植酸酶性质测定
1)植酸酶酶学性质的测定方法
有目的植酸酶的收集管用与测定植酸酶酶活，酶活测定方法如下：将上清用缓冲液(50mM Tris-HCl，2mM CaCl2，10％甘油，pH710)做适当稀释后，取015ml酶液，加入2ml 2mM的植酸钠(100mM Tris-HCl，pH710，012mM CaCl2)，37℃保温30min，加入215ml 20％TCA(w/v)终止酶活反应，5000r/min离心5min，然后加入5ml硫酸亚铁-钼酸铵显色液[(115％钼酸铵∶515％硫酸)∶(217％硫酸亚铁)＝4∶1，v/v]，5000r/min离心5min，700mM测定无机磷酸含量。对照为先在015ml的酶稀释液中加入215ml20％TCA使酶失活，再加入同体积的底物保温。酶活性单位(U)定义为：在一定条件下，每分钟释放1nmol无机磷所需的酶量为一个酶活性单位。
2)环化植酸酶和野生型植酸酶的热稳定性测定比较
植酸酶的最适温度的测定在Tris-HCl(pH 7.0)缓冲液体系及不同温度下进行酶促反应。热稳定性测定为植酸酶在不同温度下处理1小时，在37℃下进行酶活测定。酶反应最适温度测定结果(图4)表明，其最适温度为58℃。酶的热稳定性实验表明(图3)，60℃处理1小时后，剩余酶活为65％，70℃处理1小时，酶活基本丧失。而野生型植酸酶，60℃处理1小时后，剩余酶活为39％，65℃处理1小时，剩余酶活为20％，70℃处理1小时，酶活基本丧失。
3)环化植酸酶和野生型植酸酶的外肽酶抗性测定比较
环化植酸酶和野生型植酸酶的外肽酶抗性测定在Tris-HCl (pH 7.0)缓冲液体系，加入羧肽酶Y，25℃处理12小时。测定结果(图5)表明，25℃处理12小时后，环化植酸酶没有发生降解，而野生型植酸酶受外肽酶作用，发生了部分降解。实验说明环化的植酸酶对外肽酶降解有明显的抗性。
序列表说明
SEQ ID NO：1是环化酶多肽序列的的前5个氨基酸
SEQ ID NO：2是环化酶多肽序列的最后5个氨基酸
SEQ ID NO：3是枯草芽孢杆菌(Bacillus subtilis)的中性植酸酶基因phyc的基因序列。
SEQ ID NO：4是dnaE的基因序列
SEQ ID NO：5是从SEQ ID NO：3推导出的phyc编码的氨基酸序列。
序列表
<110>中国农业科学院生物技术研究所
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cttcattcct ataataccgg gaagctgaac aatgtcgata tccgttatga ttttccgttg 300
aacggaaaaa aagtcgatat cgcggcagca tccaatcgct ctgaaggaaa aaataccatt 360
gatatttacg ccattgacgg gaaaaacggc acattgcaaa gcattacaga tccagaccat 420
ccgattgcat cggcaattaa tgaggtatac ggttttacct tataccacag tcaaaaaaca 480
ggaaaatatt acgcgatggt gacagggaaa gagggtgaat ttgaacaata cgaattaaag 540
gcggacaaaa atggatacat atccggcaaa aaggtacggg cgtttaaaat gaattcccag 600
acggaaggga tggcagcaga cgatgaatac ggcaggcttt atatcgcaga agaagatgag 660
gccatttgga agttcagcgc cgagccggac ggcggcagta acggaacagt tatcgaccat 720
gccgacggca ggcatttaac tcctgatatt gaaggattga cgatttacta cgctgctgac 780
gggaaaggct atctgatggc atcaagccag ggaaacagca gctacgccat ttatgacaga 840
aaaggacaga acaaatatgt tgcggatttt cgcataacag atggtcctga aacagacggc 900
acaagcgata cagacggaat tgacgttctg ggtttcggac ttgggcctga atatccgttc 960
ggtatttttg tcgcacagga cggtgaaaat atagatcacg gccaaaaggc caatcaaaat 1020
cttaaaatcg tgccgtggga aagaattgct gatcaaatcg gcttccgccc gctggcaaat 1080
aaacaggttg acccgagaaa actgaccgac agaagcggaa aactggttcg gcgtggtagc 1140
atcgataacc tcgggatcga gggaaggggt acgctcgaga aatttgctga atat 1194
<210>4
<211>477
<212>DNA
<213>集胞蓝细菌(Synechocystis species PCC6803)
<400>4
atggttaaag ttatcggtcg tagatctctg ggcgtgcagc gcatctttga tatcggtctg 60
ccgcaggacc ataactttct gctagccaac ggcgctatcg cagctaactg cctgtctttt 120
ggtaccgaaa ttttaaccgt tgagtacggc ccattgccca ttggcaaaat tgtgagtgaa 180
gaaattaatt gttctgtgta cagtgttgat ccagaaggga gagtttacac ccaggcgatc 240
gcccaatggc atgaccgggg agagcaggaa gtattggaat atgaattgga agatggttca 300
gtaatccgag ctacctctga ccaccgcttt ttaaccaccg attatcaact gttggcgatc 360
gaagaaattt ttgctaggca actggacttg ttgactttag aaaatattaa gcaaactgaa 420
gaagctcttg acaaccatcg tcttcccttt ccattacttg acgctgggac aattaaa 477
<210>5
<211>398
<212>PRT
<213>枯草芽孢杆菌(Bacillus subtilis)
<400>5
Cys Phe Asn Ile Ser Thr Gly Leu Lys Leu Lys His His His His His
1 5 10 15
His Lys Hi s Lys Leu Ser Asp Pro Tyr His Phe Thr Val Asn Ala Ala
20 25 30
Ala Glu Thr Glu Pro Val Asp Thr Ala Gly Asp Ala Ala Asp Asp Pro
35 40 45
Ala Ile Trp Leu Asp Pro Lys Thr Pro Gln Asn Ser Lys Leu Ile Thr
50 55 60
Thr Asn Lys Lys Ser Gly Leu Val Val Tyr Ser Leu Asp Gly Lys MET
65 70 75 80
Leu His Ser Tyr Asn Thr Gly Lys Leu Asn Asn Val Asp Ile Arg Tyr
85 90 95
Asp Phe Pro Leu Asn Gly Lys Lys Val Asp Ile Ala Ala Ala Ser Asn
100 105 110
Arg Ser Glu Gly Lys Asn Thr Ile Asp Ile Tyr Ala Ile Asp Gly Lys
115 120 125
Asn Gly Thr Leu Gln Ser Ile Thr Asp Pro Asp His Pro Ile Ala Ser
130 135 140
Ala Ile Asn Glu Val Tyr Gly Phe Thr Leu Tyr His Ser Gln Lys Thr
145 150 155 160
Gly Lys Tyr Tyr Ala MET Val Thr Gly Lys Glu Gly Glu Phe Glu Gln
165 170 175
Tyr Glu Leu Lys Ala Asp Lys Asn Gly Tyr Ile Ser Gly Lys Lys Val
180 185 190
Arg Ala Phe Lys MET Asn Ser Gln Thr Glu Gly MET Ala Ala Asp Asp
195 200 205
Glu Tyr Gly Arg Leu Tyr Ile Ala Glu Glu Asp Glu Ala Ile Tr pLys
210 215 220
Phe Ser Ala Glu Pro Asp Gly Gly Ser Asn Gly Thr Val Ile Asp His
225 230 235 240
Ala Asp Gly Arg His Leu Thr Pro Asp Ile Glu Gly Leu Thr Ile Tyr
245 250 255
Tyr Ala Ala Asp Gly Lys Gly Tyr Leu MET Ala Ser Ser Gln Gly Asn
260 265 270
Ser Ser Tyr Ala Ile Tyr Asp Arg Lys Gly Gln Asn Lys Tyr Val Ala
275 280 285
Asp Phe Arg Ile Thr Asp Gly Pro Glu Thr Asp Gly Thr Ser Asp Thr
290 295 300
Asp Gly Ile Asp Val Leu Gly Phe Gly Leu Gly Pro Glu Tyr Pro Phe
305 310 315 320
Gly Ile Phe Val Ala Gln Asp Gly Glu Asn Ile Asp His Gly Gln Lys
325 330 335
Ala Asn Gln Asn Leu Lys Ile Val Pro Trp Glu Arg Ile Ala Asp Gln
340 345 350
Ile Gly Phe Arg Pro Leu Ala Asn Lys Gln Val Asp Pro Arg Lys Leu
355 360 365
Thr Asp Arg Ser Gly Lys Leu Val Arg Arg Gly Ser Ile Asp Asn Leu
370 375 380
Gly Ile Glu Gly Arg Gly Thr Leu Glu Lys Phe Ala Glu Tyr
385 390 395
Claims (6)
1.一种环化的植酸酶，其多肽序列的前5个氨基酸具有SEQ ID NO：1序列，最后5个氨基酸具有SEQ ID NO：2序列；且酶蛋白的N、C两端相链接；其特征在于编码该酶的基因的核苷酸序列如SEQ ID NO：3所示。
2.权利要求1所述环化的酶，所述酶蛋白的N、C两端相链接是通过内含肽介导的环化方法，用天然的肽键将酶蛋白的N、C两端链接起来。
3.一种重组大肠杆菌细胞，含有SEQ ID NO：3所示核苷酸序列的基因。
4.一种环化酶蛋白的方法，包括表达载体的构建、重组子的筛选及环化分子的鉴定方法，其特征在于将编码权利要求1所述多肽的基因在大肠杆菌中的环化表达。
5.权利要求4所述的环化酶蛋白的方法，是利用天然集胞蓝细菌(Synechocystis species)PCC6803Ssp DnaE内含肽的转剪接特性，分别于体内体外对蛋白质环化。
6.权利要求4或5所述的环化酶蛋白的方法，在大肠杆菌中的环化表达的基因是SEQ ID NO：3所示序列的基因。
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Citations (1)
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| CN1354788A (zh) * | 1998-12-18 | 2002-06-19 | 宾州研究基金会 | 环肽 |
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| CN1354788A (zh) * | 1998-12-18 | 2002-06-19 | 宾州研究基金会 | 环肽 |
Non-Patent Citations (5)
| Title |
|---|
| Akira Kanno 等.Cyclic luciferase for Real-Time Sensing of Caspase-3 Activities in Living Mammals.《Angewandte Chemie》.2007,第119卷(第40期),图b. * |
| 刘梅英 等.从后处理工艺提高植酸酶热稳定性的研究进展.《饲料工业》.2007,第28卷(第11期),2-4. * |
| 李素丽 等.蛋白质内含肽及其生物学意义.《生物技术通讯》.2005,第16卷(第5期),552-555. * |
| 梁如冰.大肠杆菌条件性基因表达调控系统的构建与应用.《中国博士学位论文(基础科学辑)》.2007,(第4期),全文. * |
| 郝岗平，杨清.蛋白质内含肽的研究进展.《生命科学研究》.2002,第6卷(第4期),136-139. * |
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