专利名称：肝素酶i融合蛋白的制作方法肝素酶，也称肝素裂解酶，是目前已知仅有的可降解肝素的酶。肝素酶最早是在肝素黄杆菌Flavobacterium Heparinum中发现的,共有三种,分别为肝素酶I (EC 4.2.2.7)、肝素酶II (EC unassigned)、肝素酶III (EC 4.2.2.8)。其中，肝素酶I以肝素为主要底物，肝素酶II以肝素和硫酸乙酰肝素为底物，而肝素酶III以硫酸乙酰肝素为主要底物肝素酶具有广泛而重要的用途。通过肝素酶选择性降解可以有助于研究多糖的结构和序列；肝素酶可以用于临床手术后血液的去肝素化；肝素酶可以用于制备低分子量肝素。其中，肝素酶I是制备低分子量肝素的主要酶。目前，已发现肝素酶I在多种微生物中都有表达，包括肝素黄杆菌Flavobacterium Heparinum(Ram Sasisekharan et al., Cloning and expressionof heparinase I gene from Flavobacterium heparinum.Proc.Natl.Acad.Sci1993 vol.90:3660-3664)、类便拟杆菌(Wan-Seok Kim et al.，Purification andCharacterization of Heparin Lyase I from Bacteroides stercoris HJ-15.J.Biochem.Mol.Biol 2000，vol.37:684-690)、多形拟杆菌(Yongde Luo et al.，High yield, purityand activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinaseI from Escherichia col1.Arch Biochem Biophys 2007，vol.460:17-24)等。肝素酶 I 可以从这些微生物的发酵液中提取，但产量很低，且通常需要多步色谱纯化，又进一步降低了得率。因此，釆用天然提取的方法制备肝素酶I成本很高，限制了肝素酶I的工业化应用。 Ram Sasiekharan 等(Ram Sasisekharan et al.，Cloning and expressionof heparinase I gene from Flavobacterium heparinum.Proc.Natl.Acad.Sci I993vol.90:3660-3664)首先从肝素黄杆菌中克隆了肝素酶I基因，并在大肠杆菌中重组表达，证明重组肝素酶I具有与天然肝素酶I相同的活性。Steffen Ernst等(SteffenErnst et al., Expression in Escherichia coli，purification and characterizationof heparinase I from Flavobacterium heparinum,Biochem.J.1996,vol 315:589-597)利用pET系统在大肠杆菌中实现了肝素酶I的高效表达，但绝大部分产物形成包涵体，需要经过变复性才能得到有活性的蛋白。融合表达已被证明是一种增加重组蛋白可溶性的有效手段。将一些伴侣基因通过基因工程手段融合到目的基因的N端，可极大提高目的蛋白的可溶性。已发现的融合伴侣包括谷胱甘妝转移酶 GST (P.A.Nygren et al, Engineering proteins to facilitatebioprocessing, Trends Biotechnol.1994vol.12:184-188)、硫氧还蛋白 Trx (A1-LongSun et al.， Fusion expression of human pro-urokinase with E.coli thioredoxin，Biochemistry And Molecular Biology International 1998, vol 46:479-486)、麦芽糖结合蛋白 MBP (S.Nallamsetty et al., Solubility-enhancing proteins MBP and NusA playa passive role in the folding of their fusion partners,Protein Expr.Purif 2006，vol.45:175-182)、小分子泛素样修饰蛋白 SUM0(J.G.Marblestone et al.,Comparison ofSUMO fusion technology with traditional gene fusion systems !enhanced expressionand solubility with SUM0.Protein Sc1.2006，vol.15:182-189)、翻译起始因子 2IF2 (H.P.Sorensen et al.，A favorable solubility partner for the recombinant expressionof streptavidin.Protein Expr.Purif.2003, vol.32:252-259)等。目前，已有报道，通过与 GST(Yongde Luo et al.,High yield,purity and activity of soluble recombinantBacteroides thetaiotaomicron GST-heparinase I from Escherichia col1.ArchBiochem Biophys 2007，vol.460:17-24)和 MBP(Yin Chen et al.，Construction ofrecombinant Escherichia coli for over-production of soluble heparinase I byfusion to maltose-binding protein.Biochemical Engineering Journal2005，vol.23:155-159)融合可实现肝素酶I的可溶性表达，且肝素酶融合蛋白均有活性。但这些融合伴侣分子量较大(GST 27kD，MBP 40kD，肝素酶I 42kD)，使得在相同表达量下，活性区域即肝素酶I所占的比例不高，使得融合蛋白的比活降低；且较大的融合伴侣会对肝素酶I产生空间位阻，进一步影响融合蛋白的活性(Yin Chen et al.,Production of MBP-HepA fusionprotein in recombinant Escherichia coli by optimization of culture medium.Biochemical Engineering Journal2007，vol.34:114-121)。因此，减小融合伴侣的分子量，降低其空间位阻，提高肝素酶I融 合蛋白的比活，具有重要的工业化意义。
本发明提供一种或几种肝素酶I融合蛋白及其编码基因。肝素酶I融合蛋白包含三个结构域:融合结构域、连接结构域和肝素酶I结构域。其中，融合结构域是硫氧还蛋白Trx、小分子泛素样修饰蛋白SUMO和翻译起始因子2IF2其中的一种，肝素酶I结构域包括肝素黄杆菌肝素酶I。本发明还提供上述肝素酶I融合蛋白的表达、纯化方法。本发明提供了一种肝素酶I融合蛋白，包含三个结构域:融合结构域、连接结构域和肝素酶I结构域，所述的融合结构域包括硫氧还蛋白(Trx)、小分子泛素样修饰蛋白(SUMO)和翻译起始因子2(IF2)，肝素酶I结构域包括肝素黄杆菌肝素酶I。所述的硫氧还蛋白(Trx)的编码基因是(SEQ ID N0.12);小分子泛素样修饰蛋白(SUMO)的编码基因是(SEQ ID N0.13);翻译起始因子2(IF2)的编码基因是(SEQ IDN0.14);所述的连接结构域的编码基因是(SEQ ID N0.15);并且所述的肝素黄杆菌肝素酶I的编码基因是(SEQ ID N0.16)。并且所述Trx-FH heparinase I 的编码基因是(SEQ ID N0.17),SUMO-FHheparinase I 的编码基因是(SEQ ID N0.18)，IF2-FH heparinase I 的编码基因是(SEQ ID N0.19)。其中硫氧还蛋白(Trx)的氨基酸序列是:Met Ser Asp Lys He He His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val LeuLys AlaAsp Gly Ala He Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys MetHe AlaPro He Leu Asp Glu He Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala LysLeu AsnIle Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly He Arg Gly He Pro ThrLeu LeuLeu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys GlyGln LeuLys Glu Phe Leu Asp Ala Asn Leu Ala小分子泛素样修饰蛋白(SUMO)的氨基酸序列是:Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu ValLys ProGlu Thr His He Asn Leu Lys Val Ser Asp Gly Ser Ser Glu lie Phe Phe Lyslie Lys LysThr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly LysGlu Met AspSer Leu Arg Phe Leu Tyr Asp Gly lie Arg lie Gln Ala Asp Gln Thr ProGlu Asp LeuAsp Met Glu Asp Asn Asp lie lie Glu Ala His Arg Glu Gln lie Gly Gly翻译起始因子2(IF2)的编码基因是:Met Thr Asp Val Thr He Lys Thr Leu Ala Ala Glu Arg Gln Thr Ser Val GluArgLeu Val Gln Gln Phe Ala Asp Ala Gly lie Arg Lys Ser Ala Asp Asp Ser Val SerAlaGln Glu Lys Gln Thr Leu lie Asp His Leu Asn Gln Lys Asn Ser Gly Pro Asp LysLeuThr Leu Gln Arg Lys Thr Arg Ser Thr Leu Asn lie Pro Gly Thr Gly Gly Lys SerLysSer Val Gln lie Glu Val Arg Lys Lys Arg Thr Phe Val Lys Arg Asp Pro Gln GluAlaGlu Arg Leu Ala Ala Glu Glu Gln Ala Gln Arg Glu Ala Glu Glu Gln Ala Arg ArgGluAla Glu Glu Ser Ala Lys Arg Glu Ala Gln Gln Lys Ala Glu Arg Glu Ala Ala GluGlnAla Lys Arg Glu Ala Ala Glu Gln Ala Lys Arg Glu Ala Ala Glu Lys Asp Lys Val肝素黄杆菌肝素酶I的氨基酸序列是:Gln Gln Lys Lys Ser Gly Asn He Pro Tyr Arg Val Asn Val Gln Ala Asp SerAlaLys Gln Lys Ala Ile He Asp Asn Lys Trp Val Ala Val Gly He Asn Lys Pro TyrAla LeuGln Tyr Asp Asp Lys Leu Arg Phe Asn Gly Lys Pro Ser Tyr Arg Phe Glu LeuLys AlaGlu Asp Asn Ser Leu Glu Gly Tyr Ala Ala Gly Glu Thr Lys Gly Arg Thr GluLeu SerTyr Ser Tyr Ala Thr Thr Asn Asp Phe Lys Lys Phe Pro Pro Ser Val Tyr GlnAsn AlaGln Lys Leu Lys Thr Val Tyr His Tyr Gly Lys Gly He Cys Glu Gln Gly SerSer ArgSer Tyr Thr Phe Ser Val Tyr He Pro Ser Ser Phe Pro Asp Asn Ala Thr ThrHe Phe AlaGln Trp His Gly Ala Pro Ser Arg Thr Leu Val Ala Thr Pro Glu Gly GluHe Lys ThrLeu Ser He Glu Glu Phe Leu Ala Leu Tyr Asp Arg Met He Phe Lys LysAsn He AlaHis Asp Lys Val Glu Lys Lys Asp Lys Asp Gly Lys He Thr Tyr Val AlaGly Lys ProAsn Gly Trp Lys Val Glu Gln Gly Gly Tyr Pro Thr Leu Ala Phe Gly PheSer Lys GlyTyr Phe Tyr He Lys Ala Asn Ser Asp Arg Gln Trp Leu Thr Asp Lys AlaAsp Arg AsnAsn Ala Asn Pro Glu Asn Ser Glu Val Met Lys Pro Tyr Ser Ser Glu TyrLys Thr SerThr He Ala Tyr Lys Met Pro Phe Ala Gln Phe Pro Lys Asp Cys Trp IleThr Phe AspVal Ala He Asp Trp Thr Lys Tyr Gly Lys Glu Ala Asn Thr He Leu LysPro Gly LysLeu Asp Val Met Met Thr Tyr Thr Lys Asn Lys Lys Pro Gln Lys Ala HisHe Val AsnGln Gln Glu He Leu He Gly Arg Asn Asp Asp Asp Gly Tyr Tyr Phe LysPhe Gly IleTyr Arg Val Gly Asn Ser Thr Val Pro Val Thr Tyr Asn Leu Ser Gly TyrSer Glu ThrAla Arg并且所述Trx-FH heparinase I的氨基酸序列是: Met Ser Asp Lys He He His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val LeuLys AlaAsp Gly Ala lie Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Metlie AlaPro He Leu Asp Glu lie Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala LysLeu AsnIle Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly lie Arg Gly lie Pro ThrLeu LeuLeu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys GlyGln LeuLys Glu Phe Leu Asp Ala Asn Leu Ala Thr Ser Gly Gly Gly Gly Ser Gly GlyGly GlySer Asp Asp Asp Asp Lys Ala Gln Gln Lys Lys Ser Gly Asn lie Pro Tyr ArgVal AsnVal Gln Ala Asp Ser Ala Lys Gln Lys Ala lie lie Asp Asn Lys Trp Val AlaVal Gly IleAsn Lys Pro Tyr Ala Leu Gln Tyr Asp Asp Lys Leu Arg Phe Asn Gly LysPro Ser TyrArg Phe Glu Leu Lys Ala Glu Asp Asn Ser Leu Glu Gly Tyr Ala Ala GlyGlu Thr LysGly Arg Thr Glu Leu Ser Tyr Ser Tyr Ala Thr Thr Asn Asp Phe Lys LysPhe Pro ProSer Val Tyr Gln Asn Ala Gln Lys Leu Lys Thr Val Tyr His Tyr Gly LysGly He CysGlu Gln Gly Ser Ser Arg Ser Tyr Thr Phe Ser Val Tyr lie Pro Ser SerPhe Pro Asp AsnAla Thr Thr lie Phe Ala Gln Trp His Gly Ala Pro Ser Arg Thr LeuVal Ala Thr ProGlu Gly Glu lie Lys Thr Leu Ser lie Glu Glu Phe Leu Ala Leu TyrAsp Arg Met IlePhe Lys Lys Asn lie Ala His Asp Lys Val Glu Lys Lys Asp Lys AspGly Lys lie ThrTyr Val Ala Gly Lys Pro Asn Gly Trp Lys Val Glu Gln Gly Gly TyrPro Thr Leu AlaPhe Gly Phe Ser Lys Gly Tyr Phe Tyr lie Lys Ala Asn Ser Asp ArgGln Trp Leu ThrAsp Lys Ala Asp Arg Asn Asn Ala Asn Pro Glu Asn Ser Glu Val MetLys Pro Tyr SerSer Glu Tyr Lys Thr Ser Thr lie Ala Tyr Lys Met Pro Phe Ala GlnPhe Pro Lys AspCys Trp lie Thr Phe Asp Val Ala He Asp Trp Thr Lys Tyr Gly LysGlu Ala Asn Thr IleLeu Lys Pro Gly Lys Leu Asp Val Met Met Thr Tyr Thr Lys AsnLys Lys Pro Gln LysAla His lie Val Asn Gln Gln Glu He Leu lie Gly Arg Asn AspAsp Asp Gly Tyr TyrPhe Lys Phe Gly lie Tyr Arg Val Gly Asn Ser Thr Val Pro ValThr Tyr Asn Leu SerGly Tyr Ser Glu Thr Ala ArgSUMO-FH heparinase I 氨基酸序列是:Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu ValLys ProGlu Thr His lie Asn Leu Lys Val Ser Asp Gly Ser Ser Glu lie Phe Phe Lyslie Lys LysThr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly LysGlu Met AspSer Leu Arg Phe Leu Tyr Asp Gly lie Arg lie Gln Ala Asp Gln Thr ProGlu Asp LeuAsp Met Glu Asp Asn Asp lie lie Glu Ala His Arg Glu Gln lie Gly GlyThr Ser GlyGly Gly Gly Ser Gly Gly Gly Gly Ser Asp Asp Asp Asp Lys Ala Gln GlnLys Lys SerGly Asn lie Pro Tyr Arg Val Asn Val Gln Ala Asp Ser Ala Lys Gln LysAla lie He AspAsn Lys Trp Val Ala Val Gly He Asn Lys Pro Tyr Ala Leu Gln TyrAsp Asp Lys LeuArg Phe Asn Gly Lys Pro Ser Tyr Arg Phe Glu Leu Lys Ala Glu AspAsn Ser Leu GluGly Tyr Ala Ala Gly Glu Thr Lys Gly Arg Thr Glu Leu Ser Tyr SerTyr Ala Thr ThrAsn Asp Phe Lys Lys Phe Pro Pro Ser Val Tyr Gln Asn Ala Gln LysLeu Lys Thr ValTyr His Tyr Gly Lys Gly He Cys Glu Gln Gly Ser Ser Arg Ser TyrThr Phe Ser Val TyrIle Pro Ser Ser Phe Pro Asp Asn Ala Thr Thr He Phe Ala GlnTrp His Gly Ala Pro SerArg Thr Leu Val Ala Thr Pro Glu Gly Glu lie Lys Thr Leu
本发明提供一种或几种肝素酶I融合蛋白及其编码基因。肝素酶I融合蛋白包含三个结构域融合结构域、连接结构域和肝素酶I结构域。其中，融合结构域选自硫氧还蛋白Trx、小分子泛素样修饰蛋白SUMO或翻译起始因子2IF2，肝素酶I结构域包括肝素黄杆菌肝素酶I。本发明还提供上述肝素酶I融合蛋白的表达、纯化方法。