CN101525628A - 在原核宿主细胞中制备重组蛋白 - Google Patents
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Abstract
本发明涉及在原核宿主细胞中制备异源蛋白的便利方法,是通过改进所述宿主细胞中密码子的应用和/或编码罕有密码子的tRNA的表达进行的。
Description
本申请是中国申请01813410.6的分案申请,该母案于2001年7月21日以国际申请号PCT/EP01/08660提交,于2003年01月27日进入中国国家阶段,本分案采用了与该母案一致的发明名称。
技术领域
本发明涉及在原核宿主细胞中制备异源蛋白的便利方法,是通过在所述的宿主细胞中改进密码子的应用和/或编码罕有密码子(rarely occurringcodon)的tRNA的表达而进行的。
背景技术
许多细菌,尤其是革兰氏阳性细菌,已经用作宿主细胞在不含内毒素的环境里来制备异源重组蛋白,例如分泌性蛋白(参考文献8,9,19)。例如,肉葡萄球菌(Staphylococcus carnosus)这种在食品工业中用于肉类和鱼的发酵的细菌是合适上述目的的细菌。其上清中不含毒力因子和蛋白水解活性,能分泌大量的蛋白质(10)。而且,仅分泌少量的由宿主细胞编码的蛋白质,这使得较容易纯化所产生的蛋白质。例如,细菌酶(7,4,17)已经能重组产生并表达在肉葡萄球菌中。那些在其表面表达诸如链球菌蛋白G的重组肉葡萄球菌菌株尤其可作为活疫苗应用(5,12)。
许多细菌系统的一个关键的不利因素是它们对罕有密码子的应用,这些密码子与人类基因中优选的密码子有很大差异。大肠杆菌中罕有密码子的存在导致延迟和降低重组基因的表达(2,6)。所以本发明的问题是克服现有技术的这种不利之处,并提供具有改善的性质的原核表达系统。
发明内容
上述问题在本发明的权利要求书和说明书范围内得以解决。
本发明并不意欲以任何方式对权利要求书和说明书中的单个或多个应用进行限制,所述的应用还包括其替代形式。
本发明涉及在原核宿主细胞中制备异源重组蛋白的方法,其特征在于测定宿主细胞对宿主细胞基因的密码子应用,在宿主细胞中编码异源重组蛋白的核酸中,罕有密码子被常见密码子(frequent codon)所取代,所述宿主细胞用编码该重组蛋白的核酸转化,并且表达该重组核酸。与本领域已知的方法相比,用本发明的方法有可能获得该异源蛋白的明显更好的表达率。在细胞壁上的共价锚定依赖于完整的C-末端,而本发明的方法不会产生缺少完整C-末端的分泌型截短蛋白,因此本发明的方法还尤其有利于在原核生物表面表达重组蛋白。
异源的意思是所述原核宿主细胞中的所述蛋白质并不是天然的,即它对于宿主细胞而言是罕见的蛋白质。“重组体”是指由分子生物学方法产生。异源重组蛋白可以是本领域技术人员所熟知的任何蛋白质,例如,胰岛素,hGH,tPA,细胞因子,例如白细胞介素(IL)诸如IL-1,IL-2,IL-3,IL-4,IL-5,IL-6,IL-7,IL-8,IL-9,IL-10,IL-11,IL-12,IL-13,IL-14,IL-15,IL-16,IL-17,IL-18,干扰素(IFN)α,IFNβ,IFNγ,IFNω,或IFN tau,肿瘤坏死因子(TNF),TNFα和TNFβ,TRAIL;G-CSF,GM-CSF,M-CSF,MCP-1和VEGF。原核宿主细胞可以是技术人员所熟知的任何宿主细胞,尤其是革兰氏阳性和革兰氏阴性宿主细胞。这些可以是例如,大肠杆菌,枯草芽孢杆菌,链霉菌属,奇异变形菌或优选葡萄球菌属,例如特别是肉葡萄球菌,例如可从公共保藏单位,例如德意志微生物和细胞培养物保藏中心,Braunschweig,德国,得到的菌株TM300(DSM 4600,4601或4602)。
在本发明方法的范围内,首要的是对宿主细胞中尽可能多的天然蛋白在基本编码基因序列中的密码子(即编码的核苷酸三联体)应用进行分析。通过实施例分析宿主生物肉葡萄球菌菌株TM300(10)的51个基因的密码子频率,并在下面表1中显示:
表1
发现葡萄球菌(Staphylococci)的编码序列例如具有约30%的极低G+C含量,并且富含A+T的密码子占优势。所以本发明重组蛋白质,例如hGH的产量可通过两种方式提高:(i)将编码基因序列中的罕有密码子变换成常见密码子(参见实施例1)和/或(ii)表达罕有tRNA(tRNA=转运RNA,参见实施例2)。
本发明还涉及制备异源重组蛋白的方法,其中对于肉葡萄球菌而言如表1中所示或对于本发明所有其它的宿主细胞以相似的方式,将部分或全部罕有密码子由常见密码子取代,例如,根据表1中的分析,可将Glu密码子GAG由GAA取代或将Leu-密码子CTC,CTG和CTA用TTA,TTG或CTT取代。
本发明因此还涉及在原核宿主细胞中制备异源重组蛋白的方法,其特征在于测定宿主细胞中宿主细胞基因的密码子应用(所述宿主细胞具有编码特异于罕有密码子的一或多种tRNA的核酸),用所述编码重组蛋白的核酸来转化所述宿主细胞,使所述核酸表达(参见实施例2)。本领域技术人员通过分析基因或cDNA(或通过mRNA的反转录)可确定宿主细胞的罕有密码子。已经对例如表1中的宿主细胞肉葡萄球菌菌株TM300(10)进行了这种分析。本发明包括针对宿主细胞中一到所有个罕有tRNA而导入tRNA或其编码核酸。因所述宿主细胞携带有特异于罕有密码子的tRNA,故与使用本领域已知方法的情况相比,本发明的异源重组蛋白表达的量较大。本发明方法这一意外的有利性质也在实施例2中显示。单独使用本发明方法或者同时还用常见密码子取代宿主细胞编码异源蛋白的核酸中的罕有密码子都可能出现上述情况。
所以本发明包括优选的方法,其特征在于,宿主细胞中编码异源重组蛋白的核酸中,罕有密码子被常见密码子所取代,并且所述宿主细胞由编码罕有密码子的一或多种tRNA以及编码重组蛋白的所述核酸来转化。
本发明包括优选的方法,其特征在于宿主细胞中所用的平均频率低于15%的密码子被平均频率至少为15%的密码子所取代;优选的是其特征在于宿主细胞中所用的平均频率7-12%的密码子被平均频率大于12%的密码子所取代;最优选的是其特征在于宿主细胞中所用的平均频率最高达到7或10%的密码子被平均频率大于7或10%的密码子所取代;另一优选的实施方案是优选的本发明的方法,其特征在于宿主细胞中最不常见的一或多种的密码子被所述宿主细胞中最常见的一或多种的密码子所取代。一个例外是对于表达绝对必需的所述密码子。
本发明还包括本发明优选的方法,其特征在于宿主细胞中所用的平均频率低于10%的密码子被平均频率至少为10%的密码子所取代。
本发明进一步包括本发明优选的方法,其特征在于宿主细胞为大肠杆菌,可由公共保藏机构获得,例如德意志微生物和细胞培养物保藏中心,Braunschweig,德国,如大肠杆菌菌株K12 JM107(DSM 3950)。
本发明进一步包括本发明优选的方法,其特征在于宿主细胞选自葡萄球菌属。
本发明进一步包括本发明优选的方法,其特征在于宿主细胞是肉葡萄球菌(参见实施例1和2)。
在本发明优选的方法中,重组蛋白是抗体蛋白。抗体蛋白也指片段,例如Fab片段(“抗原结合片段=Fab”),F(ab’)2片段,Fv片段(“可变片段=可变部分的片段”),或单链Fv(scFv)),微体(minibodies),二聚体(dia-),三聚体(tria-)和四聚体(tetrabodies)。
在本发明优选的方法中,重组蛋白是胰岛素。
在本发明优选的方法中,重组蛋白是组织纤溶酶原激活物(tPa)。
在本发明优选的方法中,重组蛋白是人类蛋白。
在本发明优选的方法中,重组蛋白是生长激素。
在本发明特别优选的方法中,重组蛋白人生长激素(hGH,参见实施例1和2)。
在本发明另一优选的方法中,其特征在于富含G或C的密码子被含有A或T的密码子所取代。这意味着含有较多G或C的密码子被含有例如A或T而不是G或C的密码子所取代;这不意味着这些密码子不包含G或C(参见下述最优选实施方案)。
在本发明的方法中,所发现的所有罕有密码子被常见密码子(参见例如表1)所取代,并且不仅是下面的优选实施方案中指定的那些。
本发明另一特别优选的方法的特征在于密码子GCC由GCA取代。
本发明另一特别优选的方法的特征在于密码子TGC由TGT取代。
本发明另一特别优选的方法的特征在于密码子GAC由GAT取代。
本发明另一特别优选的方法的特征在于密码子GAG由GAA取代。
本发明另一特别优选的方法的特征在于密码子TTC由TTT取代。
本发明另一特别优选的方法的特征在于密码子GGG由GGT取代。
本发明另一特别优选的方法的特征在于密码子CAC由CAT取代。
本发明另一特别优选的方法的特征在于密码子ATA由ATT取代。
本发明另一特别优选的方法的特征在于密码子AAG由AAA取代。
本发明另一特别优选的方法的特征在于密码子CTC由TTA取代。
本发明另一特别优选的方法的特征在于密码子AAC由AAT取代。
本发明另一特别优选的方法的特征在于密码子CCC由CCA取代。
本发明另一特别优选的方法的特征在于密码子CAG由CAA取代。
本发明另一特别优选的方法的特征在于密码子CGG由CGT取代。
本发明另一特别优选的方法的特征在于密码子TCG由TCA取代。
本发明另一特别优选的方法的特征在于密码子ACC由ACA取代。
本发明另一特别优选的方法的特征在于密码子GTC由GTT取代。
本发明另一特别优选的方法的特征在于密码子TAC由TAT取代。
本发明另一特别优选的方法的特征在于密码子TGA由TAA取代。
本发明另一特别优选的方法的特征在于宿主细胞由编码AGG-tRNA或AGA-tRNA的核酸以及编码重组蛋白的核酸来转化。
本发明还特别包括编码重组异源蛋白的核酸分子,其特征在于宿主细胞中至少一个罕见的密码子被宿主细胞中常见的密码子所取代。适用于宿主细胞肉葡萄球菌的密码子的例子见表1。本发明包括核酸分子,其中对肉葡萄球菌而言或类似的对本发明所有其它宿主细胞而言,部分或全部罕有密码子被常见密码子所取代,例如,Glu密码子GAG被GAA所取代,或Leu密码子CTC,CTG和CTA被TTA,TTG或CTT所取代。
本发明包括编码重组异源蛋白的核酸分子,其特征在于宿主细胞中所用的平均频率低于15%的密码子被平均频率至少为15%的密码子所取代;优选的是其特征在于宿主细胞中所用的平均频率7-12%的密码子被平均频率大于12%的密码子所取代;最优选的是其特征在于宿主细胞中所用的平均频率最高达到7或10%的密码子被平均频率大于7或10%的密码子所取代;另一优选的实施方案是编码异源重组蛋白的核酸分子,其特征在于宿主细胞中最少见的一或多种密码子被所述宿主细胞中最多见的一或多种密码子所取代。例外的是对表达绝对必需的那些密码子。
本发明因此在优选的实施方案中涉及核酸分子,其特征在于宿主细胞中所用的平均频率低于10%的密码子被平均频率至少为10%的密码子所取代。
本发明还涉及本发明的核酸分子,其特征在于其编码人生长激素hGH。
本发明涉及核酸分子,其特征在于包括来自下述组的核苷酸序列:下述核苷酸序列
NNAGATCTAAAGGAGGTAATTCATATGAAAGAAACAAAACATCAACACACATTTTCTATCCGTAAGTCGGCTTATGGTGCCGCGTC
GGTTATGGTCGCATCATGTATATTTGTCATCGGTGGGGGCGTGGCAGAGGCAAATGATTCGACAACACAAACAACGACACCACTAG
AAGTCGCTCAAACGTCGCAGCAAGAAACACATACACATCAAACACCTGTTACATCATTACATACTGCAACACCTGAACATGTTGAT
GACTCTAAAGAAGCAACACCTTTACCTGAAAAAGCAGAGTCACCAAAAACCGAAGTGACAGTTCAACCTTCATCGCATACACAGGA
AGTACCTGCGTTACATAAAAAAACACAGCAACAACCGGCGTATAAGGATAAAACGGTACCAGAGTCAACGATAGCATCAAAGTCGG
TTGAATCAAATAAAGCAACAGAAAATGAGATGTCACCTGTTGAACATCATGCTTCAAATGTGGAAAAACGTGAAGATAGATTGGAG
ACTAATGAGACAACACCGCCATCAGTGGACCGTGAATTTAGCCATAAAATCATCAATAATACGCACGTAAATCCAAAAACGGATGG
ACAAACAAACGTTAATGTTGATACGAAAACGATAGACACCGTTTCACCGAAAGATGACAGAATAGATACGGCGCAACCGAAACAAG
TCGACGTTCCTAAAGAAAATACAACGGCACAAAATAAATTTACATCACAAGCGAGCGACAAAAAACCAACAGTAAAAGATGCATCA
GGTGGTGATGATGATGATAAATTTCCAACAATTCCCTTAAGTCGATTGTTCGATAACGCTATGTTACGTGCACATAGATTACACCA
GCTAGCATTCGATACTTATCAAGAATTTGAGGAAGCTTACATCCCAAAAGAACAAAAGTATAGCTTTTTACAAAATCCGCAAACAT
CATTATGTTTCTCTGAATCAATTCCAACACCTAGTAACCGTGAGGAAACTCAGCAAAAATCAAATTTAGAACTTTTACGTATTAGC
TTGTTACTTATACAATCTTGGTTAGAACCAGTTCAATTTTTACGTTCAGTATTCGCAAATAGTTTAGTCTATGGTGCTTCAGACTC
TAACGTATACGATTTATTGAAAGACTTAGAAGAAGGAATTCAAACATTAATGGGTCGTTTGGAAGATGGTTCACCAAGAACTGGCC
AAATTTTTAAACAAACATATAGCAAATTCGATACTAATTCACATAACGATGACGCATTACTTAAAAATTACGGTTTATTGTATTGT
TTTCGTAAAGATATGGATAAAGTTGAAACATTCTTACGCATAGTACAATGCCGTTCTGTTGAAGGATCATGTGGTTTTTAATGATA
ACTGCAG
(SEQ:ID-NO.1)或其部分序列,严谨条件下能与所述序列杂交的核酸,基于简并密码子的所述序列的等位变体或功能变体或所述核酸的变体。本发明范围内N表示本领域已知的任何核苷酸。
本发明还涉及由SEQ:ID-NO.1核苷酸序列编码的核酸分子。
NNAGATCTAAAGGAGGTAATTCATATGAAAGAAACAAAACATCAACACACATTTTCTATCCGTAAGTCGGCTTATGGTGCCGCGTC
GGTTATGGTCGCATCATGTATATTTGTCATCGGTGGGGGCGTGGCAGAGGCAAATGATTCGACAACACAAACAACGACACCACTAG
AAGTCGCTCAAACGTCGCAGCAAGAAACACATACACATCAAACACCTGTTACATCATTACATACTGCAACACCTGAACATGTTGAT
GACTCTAAAGAAGCAACACCTTTACCTGAAAAAGCAGAGTCACCAAAAACCGAAGTGACAGTTCAACCTTCATCGCATACACAGGA
AGTACCTGCGTTACATAAAAAAACACAGCAACAACCGGCGTATAAGGATAAAACGGTACCAGAGTCAACGATAGCATCAAAGTCGG
TTGAATCAAATAAAGCAACAGAAAATGAGATGTCACCTGTTGAACATCATGCTTCAAATGTGGAAAAACGTGAAGATAGATTGGAG
ACTAATGAGACAACACCGCCATCAGTGGACCGTGAATTTAGCCATAAAATCATCAATAATACGCACGTAAATCCAAAAACGGATGG
ACAAACAAACGTTAATGTTGATACGAAAACGATAGACACCGTTTCACCGAAAGATGACAGAATAGATACGGCGCAACCGAAACAAG
TCGACGTTCCTAAAGAAAATACAACGGCACAAAATAAATTTACATCACAAGCGAGCGACAAAAAACCAACAGTAAAAGATGCATCA
GGTGGTGATGATGATGATAAATTTCCAACAATTCCCTTAAGTCGATTGTTCGATAACGCTATGTTACGTGCACATAGATTACACCA
GCTAGCATTCGATACTTATCAAGAATTTGAGGAAGCTTACATCCCAAAAGAACAAAAGTATAGCTTTTTACAAAATCCGCAAACAT
CATTATGTTTCTCTGAATCAATTCCAACACCTAGTAACCGTGAGGAAACTCAGCAAAAATCAAATTTAGAACTTTTACGTATTAGC
TTGTTACTTATACAATCTTGGTTAGAACCAGTTCAATTTTTACGTTCAGTATTCGCAAATAGTTTAGTCTATGGTGCTTCAGACTC
TAACGTATACGATTTATTGAAAGACTTAGAAGAAGGAATTCAAACATTAATGGGTCGTTTGGAAGATGGTTCACCAAGAACTGGCC
AAATTTTTAAACAAACATATAGCAAATTCGATACTAATTCACATAACGATGACGCATTACTTAAAAATTACGGTTTATTGTATTGT
TTTCGTAAAGATATGGATAAAGTTGAAACATTCTTACGCATAGTACAATGCCGTTCTGTTGAAGGATCATGTGGTTTTTAATGATA
ACTGCAG
本发明上述核酸分子编码具有SEQ:ID-NO.2所示氨基酸序列的hGH。
MKETKHQHTFSIRKSAYGAASVMVASCIFVIGGGVAEANDSTTQTTTPLEVAQTSQQETHTHQTPVTSLHTATPEHVDDSKEATPL
PEKAESPKTEVTVQPSSHTQEVPALHKKTQQQPAYKDKTVPESTIASKSVESNKATENEMSPVEHHASNVEKREDRLETNETTPPS
VDREFSHKIINNTHVNPKTDGQTNVNVDTKTIDTVSPKDDRIDTAQPKQVDVPKENTTAQNKFTSQASDKKPTVKDASGGDDDDKF
PTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWL
EPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKV
ETFLRIVQCRSVEGSCGF
本发明涉及含有本发明核酸分子的载体。
本发明另一重要的实施方案是含有本发明核酸分子或本发明载体的宿主细胞。
本发明另一重要的实施方案是含有一或多种tRNA分子的宿主细胞,所述分子在该宿主细胞中编码罕有密码子。
优选葡萄球菌属的宿主细胞,最优选肉葡萄球菌,例如菌株TM300(DSM4600,4601或4602)。
下述实施例意欲使本发明更易理解,并不意欲以任何方式限定本发明的范围。
实施例1:应用肉葡萄球菌特异性密码子构建合成型hGH基因
在肉葡萄球菌菌株TM300中表达蛋白,如人生长激素(hGH)。
成熟hGH的密码子应用与肉葡萄球菌中所示的比较显示,51个成熟hGH密码子(占密码子总数的27%)在肉葡萄球菌中的密码子频率仅为7%或更低(表2)。在成熟hGH DNA序列的5’末端引入肠激酶切割位点,并根据肉葡萄球菌特异性密码子使用特点合成hGH。合成基因中所用密码子的频率约与肉葡萄球菌基因中的相同。肉葡萄球菌中利用频率低于10%的密码子没有被应用,但引入限制性切割位点所必须的那些除外(参见表2)。
表2本发明的hGH cDNA以及合成的hGH DNA中的密码子数
1hGH cDNA中的密码子数
2本发明合成的hCG DNA中的密码子数
合成型hGH DNA序列自体外已杂交和连接的重叠寡核苷酸合成。将所得DNA片段克隆入大肠杆菌质粒pSL1190(3),通过测序证实并将其插入hGH表达盒以取代Nsi I和Pst I切割位点间的hGH cDNA(图1)。新的表达盒被分离为Bgl II-Cla I片段,克隆入葡萄球菌属-表达载体pTX15中适合的Bam HI和Nar I切割位点,通过原生质体转化方法将其转化至肉葡萄球菌TM300中。在所得表达载体中,lip-hGH融合基因在pTX15的xyl启动子控制下表达。通过向营养培养基中添加0.5%的木糖,灭活xyl启动子抑制物xylR之后,获得表达。用标准方法实施体外克隆和测序(1)。hGH基因表达为与Lip-信号和来自猪葡萄球菌的前肽的融合体,其包含肠激酶切割位点,可以自融合蛋白释放具有正确N-末端的成熟hGH(图1)。所得表达载体pTX-LipP-hGH4除了应用成熟hGH的密码子外与pTX-LipP-hGH2相同。含有其中一种质粒的肉葡萄球菌菌株在添加有0.5%木糖的改良型LB培养基(1%大豆提取物,0.5%的酵母提取物,0.5%NaCl)上培养,以激活表达载体的xyl启动子。应用改进的hGH DNA对肉葡萄球菌hGH产生的效率有深刻影响。Lip-hGH的产量至少改进两倍,并且缩短的Lip-hGH蛋白的量显著降低(图2,泳道2和3)。由于Lip-hGH蛋白在肉葡萄球菌上清中稳定存在,所以缩短的Lip-hGH蛋白不是胞外蛋白水解引起的。肉葡萄球菌上清中缩短蛋白(pTX-LipP-hGH4)的减少表明,最优化的DNA序列翻译hGH的速度更快,因此在该蛋白经细胞质膜分泌出去之前胞内蛋白酶切割该蛋白的机会较少。
实施例2:特异于hGH cDNA中罕有密码子的t-RNA的共表达
在肉葡萄球菌基因中精氨酸密码子AGG非常少见它仅使用0.8%的精氨酸密码子。肉葡萄球菌中相应的tRNA基因也未鉴定出。因此在肉葡萄球菌中表达AGG密码子的大肠杆菌tRNA。大肠杆菌基因argU和argW编码密码子AGG/AGA和AGG的tRNA,将它们与其天然启动子和终止子一起克隆入质粒pUBS520或pSB101(2,16)。分离出片段SalI-SpHI(argU)和XmaI-EcoRI(argW),并克隆入穿梭(转移)载体pRB572的相应限制切割位点(4)。将所得质粒pRBargU和pRBargW转化入肉葡萄球菌(pTX-LipP-hGH2),其表达hGH cDNA。
所得肉葡萄球菌菌株上清中的蛋白质模式与肉葡萄球菌(pTX-LipP-hGH4)中的相当,但Lip-hGH蛋白的产量增加,而截短型蛋白较少。所以,本发明罕有tRNA的表达有利于在诸如肉葡萄球菌中制备人蛋白。
附图说明
图1.表达质粒pTX-LipP-hGH2或pTX-LipP-hGH4中的Lip-hGH融合体。
基因融合是由编码来自猪葡萄球菌脂酶的信号肽(SP)和前肽(PP)的DNA序列和成熟蛋白的hGH部分(黑区)组成,由xyl启动子(用灰色三角表示)转录。hGH cDNA或合成型DNA序列在Nsi I和Pst I切割位点插入。修饰hGH DNA序列的5’末端以编码肠激酶切割位点“DDDDK”,其后接成熟hGH序列,如在该基因之下所示。
图2.检测肉葡萄球菌上清中的Lip-hGH融合蛋白
等量上清在含15%丙烯酰胺的Tris-甘氨酸胶上经SDS-PAGE分离,然后用考马斯蓝染色。泳道1-5为肉葡萄球菌菌株的上清,它们分别含:空载体pTX16(15),它是pTX15的衍生物(泳道1),具有改进的hGH基因的质粒pTX-LipP-hGH4(泳道2),具有hGH cDNA的pTX-LipP-hGH2(泳道3),pTX-LipP-hGH2加pRBargU(泳道4),pTX-LipP-hGH2加pRBargW(泳道5)。标准蛋白和其分子量(kDa)在左边显示。上清通过三氯乙酸沉淀来浓缩。使用现有技术中的标准方法进行SDS聚丙烯酰胺凝胶电泳。
图3.本发明的hGH(LipPhGH4,SEQ:ID-NO.3)的核苷酸和氨基酸序列
相关的限制性切割位点在其下单划线。Shine-Dalgarno序列在其下双划线。信号肽酶1和肠激酶的切割位点在其下以断线表示。仅合成BglII和NdeI位点间的区和NsiI和PstI位点间的区,并最优化。其余部分为猪葡萄球菌脂酶的信号和前肽之原始序列。
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17.Thumm,G.,and F.1997.Studies on prolysostaphin processing and characferization of the lysostaphinimmunity factor(Lif)of Staphylococcus simulans biovar staphylolyticus.Mol.Microbiol.23:1251-1265.
18.Vance,M.L.,and N.Mauras.1999.Growth hormone therapy in adults and children.New Engl.J.Med.341:1206-1216.
19.Wong,S.L.1995.Advances in the use of Bacillus subtilis for the expression and secretion of beterologousproteins.Curr.Opin.Biotechnol.6:517-522.
序列表
<110>贝林格尔·英格海姆国际有限公司(Boehringer Ingelheim International GmbH)
<120>在原核宿主细胞中制备重组蛋白
<140>美国申请号09/916,229
<141>2001-07-27
<160>3
<170>PatentIn Ver.2.1
<210>1
<211>1383
<212>DNA
<213>人(Homo sapiens)
<220>
<221>unsure
<222>(1)...(2)
<400>1
nnagatctaa aggaggtaat tcatatgaaa gaaacaaaac atcaacacac attttctatc 60
cgtaagtcgg cttatggtgc cgcgtcggtt atggtcgcat catgtatatt tgtcatcggt 120
gggggcgtgg cagaggcaaa tgattcgaca acacaaacaa cgacaccact agaagtcgct 180
caaacgtcgc agcaagaaac acatacacat caaacacctg ttacatcatt acatactgca 240
acacctgaac atgttgatga ctctaaagaa gcaacacctt tacctgaaaa agcagagtca 300
ccaaaaaccg aagtgacagt tcaaccttca tcgcatacac aggaagtacc tgcgttacat 360
aaaaaaacac agcaacaacc ggcgtataag gataaaacgg taccagagtc aacgatagca 420
tcaaagtcgg ttgaatcaaa taaagcaaca gaaaatgaga tgtcacctgt tgaacatcat 480
gcttcaaatg tggaaaaacg tgaagataga ttggagacta atgagacaac accgccatca 540
gtggaccgtg aatttagcca taaaatcatc aataatacgc acgtaaatcc aaaaacggat 600
ggacaaacaa acgttaatgt tgatacgaaa acgatagaca ccgtttcacc gaaagatgac 660
agaatagata cggcgcaacc gaaacaagtc gacgttccta aagaaaatac aacggcacaa 720
aataaattta catcacaagc gagcgacaaa aaaccaacag taaaagatgc atcaggtggt 780
gatgatgatg ataaatttcc aacaattccc ttaagtcgat tgttcgataa cgctatgtta 840
cgtgcacata gattacacca gctagcattc gatacttatc aagaatttga ggaagcttac 900
atcccaaaag aacaaaagta tagcttttta caaaatccgc aaacatcatt atgtttctct 960
gaatcaattc caacacctag taaccgtgag gaaactcagc aaaaatcaaa tttagaactt 1020
ttacgtatta gcttgttact tatacaatct tggttagaac cagttcaatt tttacgttca 1080
gtattcgcaa atagtttagt ctatggtgct tcagactcta acgtatacga tttattgaaa 1140
gacttagaag aaggaattca aacattaatg ggtcgtttgg aagatggttc accaagaact 1200
ggccaaattt ttaaacaaac atatagcaaa ttcgatacta attcacataa cgatgacgca 1260
ttacttaaaa attacggttt attgtattgt tttcgtaaag atatggataa agttgaaaca 1320
ttcttacgca tagtacaatg ccgttctgtt gaaggatcat gtggttttta atgataactg 1380
cag 1383
<210>2
<211>448
<212>PRT
<213>人(Homo sapiens)
<400>2
Met Lys Glu Thr Lys His G1n His Thr Phe Ser Ile Arg Lys Ser Ala
1 5 10 15
Tyr Gly Ala Ala Ser Val Met Val Ala Ser Cys Ile Phe Val Ile Gly
20 25 30
Gly Gly Val Ala Glu Ala Asn Asp Ser Thr Thr Gln Thr Thr Thr Pro
35 40 45
Leu Glu Val Ala Gln Thr Ser Gln Gln Glu Thr His Thr His Gln Thr
50 55 60
Pro Val Thr Ser Leu His Thr Ala Thr Pro Glu His Val Asp Asp Ser
65 70 75 80
Lys Glu Ala Thr Pro Leu Pro Glu Lys Ala Glu Ser Pro Lys Thr Glu
85 90 95
Val Thr Val Gln Pro Ser Ser His Thr Gln Glu Val Pro Ala Leu His
100 105 110
Lys Lys Thr Gln Gln Gln Pro Ala Tyr Lys Asp Lys Thr Val Pro Glu
115 120 125
Ser Thr Ile Ala Ser Lys Ser Val Glu Ser Asn Lys Ala Thr Glu Asn
130 135 140
Glu Met Ser Pro Val Glu His His Ala Ser Asn Val G1u Lys Arg Glu
145 150 155 160
Asp Arg Leu Glu Thr Asn Glu Thr Thr Pro Pro Ser Val Asp Arg Glu
165 170 175
Phe Ser His Lys Ile Ile Asn Asn Thr His Val Asn Pro Lys Thr Asp
180 185 190
Gly Gln Thr Asn Val Asn Val Asp Thr Lys Thr Ile Asp Thr Val Ser
195 200 205
Pro Lys Asp Asp Arg Ile Asp Thr Ala Gln Pro Lys Gln Val Asp Val
210 215 220
Pro Lys Glu Asn Thr Thr Ala Gln Asn Lys Phe Thr Ser Gln Ala Ser
225 230 235 240
Asp Lys Lys Pro Thr Val Lys Asp Ala Ser Gly Gly Asp Asp Asp Asp
245 250 255
Lys Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu
260 265 270
Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe
275 280 285
Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn
290 295 300
Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn
305 310 315 320
Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser
325 330 335
Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser
340 345 350
Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr
355 360 365
Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg
370 375 380
Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr
385 390 395 400
Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn
405 410 415
Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr
420 425 430
Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe
435 440 445
<210>3
<211>1383
<212>DNA
<213>人工序列
<220>
<221>unsure
<222>(1)...(2)
<223>人工序列的描述:人(Homo sapiens)
侧翼带有载体序列
<400>3
nnagatctaa aggaggtaat tcatatgaaa gaaacaaaac atcaacacac attttctatc 60
cgtaagtcgg cttatggtgc cgcgtcggtt atggtcgcat catgtatatt tgtcatcggt 120
gggggcgtgg cagaggcaaa tgattcgaca acacaaacaa cgacaccact agaagtcgct 180
caaacgtcgc agcaagaaac acatacacat caaacacctg ttacatcatt acatactgca 240
acacctgaac atgttgatga ctctaaagaa gcaacacctt tacctgaaaa agcagagtca 300
ccaaaaaccg aagtgacagt tcaaccttca tcgcatacac aggaagtacc tgcgttacat 360
aaaaaaacac agcaacaacc ggcgtataag gataaaacgg taccagagtc aacgatagca 420
tcaaagtcgg ttgaatcaaa taaagcaaca gaaaatgaga tgtcacctgt tgaacatcat 480
gcttcaaatg tggaaaaacg tgaagataga ttggagacta atgagacaac accgccatca 540
gtggaccgtg aatttagcca taaaatcatc aataatacgc acgtaaatcc aaaaacggat 600
ggacaaacaa acgttaatgt tgatacgaaa acgatagaca ccgtttcacc gaaagatgac 660
agaatagata cggcgcaacc gaaacaagtc gacgttccta aagaaaatac aacggcacaa 720
aataaattta catcacaagc gagcgacaaa aaaccaacag taaaagatgc atcaggtggt 780
gatgatgatg ataaatttcc aacaattccc ttaagtcgat tgttcgataa cgctatgtta 840
cgtgcacata gattacacca gctagcattc gatacttatc aagaatttga ggaagcttac 900
atcccaaaag aacaaaagta tagcttttta caaaatccgc aaacatcatt atgtttctct 960
gaatcaattc caacacctag taaccgtgag gaaactcagc aaaaatcaaa tttagaactt 1020
ttacgtatta gcttgttact tatacaatct tggttagaac cagttcaatt tttacgttca 1080
gtattcgcaa atagtttagt ctatggtgct tcagactcta acgtatacga tttattgaaa 1140
gacttagaag aaggaattca aacattaatg ggtcgtttgg aagatggttc accaagaact 1200
ggccaaattt ttaaacaaac atatagcaaa ttcgatacta attcacataa cgatgacgca 1260
ttacttaaaa attacggttt attgtattgt tttcgtaaag atatggataa agttgaaaca 1320
ttcttacgca tagtacaatg ccgttctgtt gaaggatcat gtggttttta atgataactg 1380
cag 1383
Claims (6)
1.在肉葡萄球菌中表达LipP-hGH融合基因的方法,其中将人生长激素(hGH)基因与猪葡萄球菌脂肪酶的lip-信号和前肽的编码序列(lipP)融合,并在LipP和hGH之间包含肠激酶切割位点以便能释放具有正确N-末端的成熟hGH,而且将在肉葡萄球菌中平均使用频率低于10%的编码hGH基因的密码子替换为平均使用频率至少为10%的密码子,和/或所述肉葡萄球菌宿主细胞用一或多种编码平均使用频率低于10%的密码子的tRNA或者所述tRNA的编码核酸来转化。
2.权利要求1的方法,其中hGH的富含G或C的密码子被含有A或T的密码子替换。
3.权利要求1或2的方法,其中肉葡萄球菌被编码AGG-tRNA或AGA-tRNA的核酸转化。
4.权利要求1-3之一的方法,其中肉葡萄球菌被大肠杆菌基因argU和argW的核酸转化,所述核酸编码密码子AGG/AGA和AGG的tRNA。
5.权利要求1-4之一的方法,其中LipP-hGH融合蛋白由包含SEQ:ID-NO.3所示序列的核酸分子编码。
6.编码LipP-hGH融合蛋白的核酸分子,其包含SEQ:ID-NO.3的序列。
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CN102286101A (zh) * | 2011-08-08 | 2011-12-21 | 常州亚当生物技术有限公司 | 抗VEGF单克隆抗体Fab片段Vasculizumab及其应用 |
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WO2001068835A2 (en) * | 2000-03-13 | 2001-09-20 | Aptagen | Method for modifying a nucleic acid |
US8551469B2 (en) | 2001-02-28 | 2013-10-08 | Superlab Far East Limited | Treatment of tumors and viral diseases with recombinant interferon alpha |
JP4124639B2 (ja) * | 2002-12-17 | 2008-07-23 | 株式会社日本触媒 | 大腸菌を用いたs−ヒドロキシニトリルリアーゼの製造方法 |
KR101329878B1 (ko) * | 2005-03-09 | 2013-11-22 | 광웬 위 | 재조합 슈퍼 화합물 인터페론의 용도 |
EP2021490B1 (en) | 2006-05-30 | 2011-09-07 | Pfenex, Inc. | Anthrax vaccine |
JP4886654B2 (ja) * | 2007-10-29 | 2012-02-29 | 日本ケミカルリサーチ株式会社 | ヒト成長ホルモンの効率化された製造方法 |
KR102064025B1 (ko) * | 2012-04-17 | 2020-01-08 | 에프. 호프만-라 로슈 아게 | 수식된 핵산을 사용하는 폴리펩티드의 발현 방법 |
CN107723287B (zh) * | 2016-08-12 | 2021-07-06 | 中国科学院天津工业生物技术研究所 | 一种增强丝蛋白生产制备的表达系统 |
CN108456703B (zh) * | 2017-02-20 | 2022-01-14 | 复旦大学 | 一种异源表达埃博霉素的方法 |
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AU618640B2 (en) * | 1988-11-11 | 1992-01-02 | Boehringer Mannheim Gmbh | Process for the expression of a recombinant gene |
JPH05284973A (ja) * | 1992-04-13 | 1993-11-02 | Kurita Water Ind Ltd | 組換プラスミドおよびこれをベクターとして用いる異種蛋白質の分泌生産方法 |
JP3556965B2 (ja) * | 1993-11-08 | 2004-08-25 | 天野エンザイム株式会社 | 糸状菌および酵母で使用可能なポリペプチド分泌発現用プラスミドおよびそれを用いたポリペプチドの製造法 |
AU6148798A (en) * | 1997-02-07 | 1998-08-26 | Vanderbilt University | Synthetic genes for recombinant mycobacterium proteins |
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WO2000044926A1 (en) * | 1999-01-27 | 2000-08-03 | Stratagene | High level expression of a heterologous protein having rare codons |
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CN102286101B (zh) * | 2011-08-08 | 2013-05-08 | 常州亚当生物技术有限公司 | 抗VEGF单克隆抗体Fab片段Vasculizumab及其应用 |
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ATE325880T1 (de) | 2006-06-15 |
WO2002010411A3 (en) | 2002-07-18 |
JP5044085B2 (ja) | 2012-10-10 |
EP1307569B1 (en) | 2006-05-10 |
DE10037111A1 (de) | 2002-02-07 |
CA2414978C (en) | 2009-02-24 |
WO2002010411A2 (en) | 2002-02-07 |
DK1307569T3 (da) | 2006-09-04 |
CN1444655A (zh) | 2003-09-24 |
AU2001277554A1 (en) | 2002-02-13 |
CA2414978A1 (en) | 2002-02-07 |
DE60119535T2 (de) | 2006-09-07 |
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