CN112226454A - 一种重组人生长激素的真核表达及其纯化方法 - Google Patents
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Abstract
一种重组人生长激素的真核表达及其纯化方法。本发明涉及一种酵母表达人生长激素及其纯化方法,通过人工合成优化后具有酵母表达偏好的可编码人生长激素的核苷酸序列,并通过分子克隆等技术获得可表达人生长激素的酵母重组子pPICZαA‑hGH‑GS115。重组子在适当的条件下发酵,人生长激素可分泌表达至发酵液中。发酵液进行固液分离,取上清然后通过切向流超滤以及离子交换柱,可以得到高纯度的重组人生长激素蛋白。
Description
技术领域
本发明属于生物工程基因领域,涉及一种重组人生长激素真核表达***及其纯化方法。
背景技术
人生长激素(Human Growth Hormone,以下简写为hGH)含有191个氨基酸残基,分子量为21.7kDa,等电点4.9,是由脑垂体前叶嗜酸性细胞分泌的一种单一肽链的蛋白质激素,是人类出生后促进生长的最重要的激素,具有调节人体生长代谢等多重功能,在人体生长发育过程中起着重要作用。hGH能够促进骨骼、内脏和全身生长,还能促进蛋白质合成,加快脂肪和矿物质代谢。hGH基因工程产品问世以来,适应症范围扩大到成人生长激素缺乏、Turner氏综合症、艾滋病消瘦、大面积创伤恢复等症状。随着临床研究的不断深入,生长激素在抗衰老、骨质疏松症、心血管疾病治疗方面也有很好的疗效。
全球生长激素市场非常巨大,目前利用DNA重组技术在大肠杆菌中表达的重组人生长激素,但是大肠杆菌重组表达外源蛋白会在蛋白的N端加入起始密码子,与天然蛋白相比多了起始氨基酸-蛋氨酸,有时候为了纯化方便,也会在蛋白质序列中加入一定的标签,应用该类生长激素后易产生抗体;因此,人们趋向于研究N-端不带Met或标签的人生长激素。用大肠杆菌生产无Met的人生长激素有两种方法,一种是在胞内表达产生Met-人生长激素后,在加工、纯化过程中用酶法除去Met分子;另一种方法是用分泌型载体,把人生长激素成熟蛋白的编码序列直接转录入高表达高分泌启动子和信号序列后面,在分泌过程中切去信号肽,使表达的人生长激素累积在细胞周质中。毕赤酵母作为低等真核生物,具有高效分泌表达的特点,同时可以利用自身信号肽的切割的特点,获得无Met的人生长激素。为了不产生抗体,需要不带标签和Metd的天然hGH相同序列,这就给hGH的表达纯化带来了困难。
发明内容
为了克服以上缺点,发明人对hGH基因在毕赤酵母表达***中的表达偏好进行了优化,构建重组子pPICZαA-hGH-GS115分泌表达hGH重组蛋白,并做了对表达hGH重组蛋白纯化的探索,发明人惊喜地发现,在该表达***中,酵母分泌表达的hGH可以被300kD孔径的超滤膜截留,通过300kD超滤膜截留后可以去除酵母分泌表达的绝大部分蛋白质并同时脱盐。再经过一步离子交换就可以得到纯度95%以上的hGH蛋白。hGH蛋白重组表达纯化具体如下。
一种含有编码hGH蛋白基因序列的pPICZαA-hGH载体,所述编码hGH蛋白基因序列如SEQ ID: 1所示,所述pPICZαA-hGH载体的序列为SEQ ID: 2所示。
一种酵母重组子pPICZαA-hGH-GS115,通过将pPICZαA-hGH载体同源重组到酵母GS115基因组上获得。
一种重组hGH蛋白的纯化方法,包括以下步骤:
1)将权利要求2所述的酵母重组子pPICZαA-hGH-GS115进行发酵培养并适时补料,用诱导甲醇诱导表达,获得含有重组hGH蛋白的发酵液;
2)发酵液的处理:将步骤1)所得的发酵液5000rpm离心5~10min,取上清,过2um滤膜,收集滤液;
3)超滤:将步骤2)得到的滤液用300kD超滤膜超滤,取截留液;
4)将步骤3)获得的截留液过Q离子交换柱,得到纯化的hGH蛋白。
作为优选,酵母重组子pPICZαA-hGH-GS115,通过以下步骤获得:将pPICZαA-hGH载体线性化后,电转入GS115毕赤酵母菌株中,获得酵母重组子pPICZαA-hGH-GS115。
作为优选,步骤1)当发酵培养至酵母湿重达到150mg/ml以上时,加入适量甲醇,诱导 36~60h。
作为优选,酵母重组子pPICZαA-hGH-GS115发酵培养条件为:温度30 ℃,pH 值为5.0,溶氧控制在25%~45%;甲醇诱导条件为:温度28 ℃,pH 值为5.6,溶氧控制在25%~45%。
作为优选,步骤3)所述超滤为切向超滤。
作为优选,步骤3)所述超滤包含以下步骤:将步骤2)得到的滤液调pH至6.9~7.5,过300kD切向超滤膜超滤,压力0.1~0.2兆帕,当截留液为原体积的1/8~1/15时,加入5~10倍体积的pH7.2~7.5的10 mmol/L 磷酸盐缓冲液,继续超滤至原体积1/8~1/15;重复3~8次。
作为优选,步骤4)具体包含以下步骤:pH7.2~7.5的10 mmol/L 磷酸盐缓冲液平衡2~3个柱体积,上样,孵育3~5 min,2~5倍柱体积洗涤液洗涤,含400 mmol/L NaCl的洗涤液洗脱,收集洗脱液, 脱盐,浓缩、干燥获得hGH重组蛋白。
本发明的有益效果:根据酵母表达的密码子偏爱,优化编码hGH基因的DNA序列,使重组的hGH蛋白更容易被酵母表达;通过300kD的切向流超滤膜包可以将本方法发酵上清液中的绝大多数小于300kD的蛋白质去掉,极大的简化了后续纯化步骤,并提高蛋白的分离效率和蛋白纯度。hGH分子量虽然只有21.7kD,但发酵得到的重组hGH可以穿透超滤管的300kD膜,却不能穿过300kD的切向流超滤膜包,这是意想不到的的。
附图说明
图1是300 kD滤膜超滤蛋白电泳图,其中M为蛋白质分子量Marker,1为300 kD的切向流滤膜超滤截留液,2为300 kD切向流滤膜超滤穿透液。箭头所示为表达的重组hGH。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1可表达hGH重组酵母菌的获得。
1、根据酵母表达密码子的偏爱,设计编码重组hGH蛋白的DNA序列SEQ ID NO:1,进行人工合成。
2、通过分子克隆技术构建含有编码hGH基因的pPICZαA-hGH重组质粒,序列如DNA序列SEQ ID NO:2所示。
3、将步骤2的pPICZαA-hGH重组质粒用限制性内切酶SacI线性化,并回收。将回收的线性化质粒电转入GS115毕赤酵母菌株中,获得重组菌pPICZαA-hGH-GS115。电转条件如下;取10μg上述线性化的pPICZαA-hGH质粒,与80μL毕赤酵母GS115感受态细胞混匀,转至0.2cm冰预冷的电转化杯中,电压1kV;电容30μF;电阻200Ω;电击4~10毫秒,加入1mL冰预冷的1mol/L的山梨醇溶液将菌体混匀,放入30摄氏度水浴锅,水浴30min,完成转化。将完成转化的酵母用选择培养基进行筛选,获得阳性克隆pPICZαA-hGH-GS115重组菌。
实施例2 重组人生长激素蛋白的发酵表达。
将筛选到的pPICZαA-hGH-GS115重组菌接种于300ml BMGY(酵母提取物10 g/L,蛋白胨20 g/L,K2HPO4 3 g/L,KH2PO4 11.8 g/L,YNB 13.4 g/L,生物素4×10-4 g/L,甘油10g/L)培养基中,30 ℃振荡培养24小时,作为种子转接于装有3 L BMGY培养基的5 L发酵罐中,温度设定为30 ℃,pH 值为5.0,溶氧控制在25%~45%,当溶氧陡然上升时,预示基础盐培养基中的甘油已耗尽,开始流加10-20 mL/h/L质量百分浓度为50 %的甘油(优选12mL/h/L),在质量百分浓度为50 %甘油中含12 mL/L微量元素PTM1(1L微量元素PTM1中含CuSO4.5H2O 6g、NaI 0.08 g、MnSO4.H2O 3 g、Na2MoO4.H2O 0.2 g、H3BO3 0.02 g、H2SO4 5 ml、CoCl2.6H2O0.5 g、ZnCl2 20 g、FeSO4.7H2O 75 g、生物素 0.2 g)维持溶氧在25%~45%。发酵液的湿菌重达到150~200 mg/mL时,停止补料,饥饿1-2小时,甘油耗尽,流加20 L甲醇诱导发酵,其中甲醇中含12 mL/L PTM1 微量元素,以速率为5 ml/L/h流加2~3 小时,然后提高速率至8ml/L/h流加进行发酵。通过调节转速、罐压和通气量使溶氧大于20%(优选25-45%),诱导发酵36~48小时。诱导温度28℃,pH 值为5~5.5。
实施例3重组人生长激素的纯化。
(1)超滤。
发酵结束后,发酵液离心分离,5000 rpm,10 min,取上清液。过2um滤膜,收集滤液。用氢氧化钠调滤液pH为6.9-7.5(优选7)用分子截留量为300kD的切向流超滤膜包进行超滤,超滤压力为0.1~0.2兆帕:当截留液体积减少为原体积的1/8~1/15(优选1/10)时,加入5~10倍体积的(优选5)ddH2O,继续超滤至原体积1/8~1/15;重复3~8次(优选5次);收集截留液。通过上述一步超滤法即可去除毕赤酵母发酵产生的大量蛋白质并同时脱盐,获得人生长激素蛋白浓缩液。
取超滤截留液即生长激素蛋白浓缩液和穿透液进行SDS-PAGE电泳,结果如图1(其中M为蛋白质分子量Marker,从下往上数第一条带分子量为15 kD,第二条带分子量为35kD;1泳道为300 kD滤膜超滤截留液;2泳道为300kD滤膜超滤穿透液;箭头所示为表达的重组hGH)所示。我们惊喜的发现,重组表达的人生长激素蛋白并不能穿透分子截流量为300kD的切向流超滤膜,但是发酵液上清中酵母本身分泌表达的大多数分子量小于300 kD的蛋白都穿透过膜,所以该步骤不仅浓缩脱盐,并且一步去除了发酵上清液中的绝大多数杂蛋白,蛋白质纯度可以达到85%以上。为后续纯化提供了方便。
(2)重组人生长激素的离子交换层析。
将人生长激素粗蛋白液进行Q柱阴离子交换层析,用3倍柱体积的洗涤液(pH7.2~7.5(优选7.4)的10mmol/L 磷酸盐缓冲液)平衡,洗涤液3~5倍体积稀释超滤截留液,上样,室温孵育3~5min,静置1min;用2~5倍体积的洗涤液进行洗涤,用5~8倍柱体积的含400mmol/L NaCl的洗涤液洗脱,收集洗脱液,将得到的洗脱液脱盐,收集浓缩液,冷冻干燥机冻干,得基因重组人生长激素。
经过超滤后的去除了发酵液中绝大多数的杂蛋白,在进行离子交换时,比直接用发酵上清液过离子交换柱,分辨率明显提高,而且仅用一步洗脱就可以获得纯度95%以上的重组rhGH蛋白。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。
序列表
<110> 杭州科迪赛生物科技有限公司
<120> 一种重组人生长激素的真核表达及其纯化方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 576
<212> DNA
<213> 人工序列(“人工序列”)
<400> 1
tttccaacta ttcctttgtc tagattgttc gataacgcta tgttgagagc tcatagattg 60
caccaattgg cttttgatac ttaccaagaa ttcgaagagg cttacatccc aaaggagcaa 120
aagtactctt tcttgcaaaa ccctcaaact tctttgtgtt tctctgaatc tattccaact 180
ccttctaaca gagaagagac tcaacaaaag tctaatttgg aattgttgag aatctctttg 240
ttgttgatcc aatcttggtt ggagccagtt caatttttga gatctgtttt cgctaactct 300
ttggtttatg gtgcttctga ttctaacgtt tacgatttgt tgaaggattt ggaagagggt 360
attcaaactt tgatgggtag attggaggat ggttctccta gaactggtca aatttttaag 420
caaacttact ctaagttcga tactaactct cataacgatg atgctttgtt gaaaaactac 480
ggtttgttgt actgtttcag aaaggatatg gataaggttg aaactttctt gagaatcgtt 540
caatgtagat ctgttgaggg ttcttgtggt ttttaa 576
<210> 2
<211> 4169
<212> DNA
<213> 人工序列(“人工序列”)
<400> 2
agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag 60
gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt 120
tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc 180
agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta 240
acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta 300
tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg 360
agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct 420
gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg 480
ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt 540
cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct 600
ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct 660
ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact 720
gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat 780
atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt 840
actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga 900
caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt 960
tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga 1020
agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga 1080
tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa 1140
tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc 1200
ttttccaact attcctttgt ctagattgtt cgataacgct atgttgagag ctcatagatt 1260
gcaccaattg gcttttgata cttaccaaga attcgaagag gcttacatcc caaaggagca 1320
aaagtactct ttcttgcaaa accctcaaac ttctttgtgt ttctctgaat ctattccaac 1380
tccttctaac agagaagaga ctcaacaaaa gtctaatttg gaattgttga gaatctcttt 1440
gttgttgatc caatcttggt tggagccagt tcaatttttg agatctgttt tcgctaactc 1500
tttggtttat ggtgcttctg attctaacgt ttacgatttg ttgaaggatt tggaagaggg 1560
tattcaaact ttgatgggta gattggagga tggttctcct agaactggtc aaatttttaa 1620
gcaaacttac tctaagttcg atactaactc tcataacgat gatgctttgt tgaaaaacta 1680
cggtttgttg tactgtttca gaaaggatat ggataaggtt gaaactttct tgagaatcgt 1740
tcaatgtaga tctgttgagg gttcttgtgg tttttaagaa gctgaattca cgtggcccag 1800
ccggccgtct cggatcggta cctcgagccg cggcggccgc cagctttcta gaacaaaaac 1860
tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt 1920
gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt 1980
gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt 2040
tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct 2100
tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg 2160
ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag 2220
tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat agcttcaaaa 2280
tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac 2340
ttcaaaacac ccaagcacag catactaaat tttccctctt tcttcctcta gggtgtcgtt 2400
aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg 2460
tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaatttt tttttttagt 2520
ttttttctct ttcagtgacc tccattgata tttaagttaa taaacggtct tcaatttctc 2580
aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg ttcattagaa 2640
agaaagcata gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata 2700
tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg 2760
ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg 2820
ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc 2880
tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg 2940
tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg 3000
acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc 3060
tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc 3120
gacggcggcc cacgggtccc aggcctcgga gatccgtccc ccttttcctt tgtcgatatc 3180
atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa 3240
aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta 3300
gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac 3360
gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta 3420
atttgcaagc tggagaccaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 3480
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 3540
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 3600
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 3660
gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 3720
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 3780
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 3840
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 3900
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 3960
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 4020
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 4080
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 4140
tcacgttaag ggattttggt catgagatc 4169
Claims (9)
1.一种含有编码hGH蛋白基因序列的pPICZαA-hGH载体,其特征在于所述编码hGH蛋白基因序列如SEQ ID: 1所示,所述pPICZαA-hGH载体的序列为SEQ ID: 2所示。
2.一种酵母重组子pPICZαA-hGH-GS115,其特征在于通过将权利要求1所述的pPICZαA-hGH载体同源重组到酵母GS115基因组上获得。
3.一种重组hGH蛋白的纯化方法,其特征在于包括以下步骤:
1)将权利要求2所述的酵母重组子pPICZαA-hGH-GS115进行发酵培养并适时补料,用诱导甲醇诱导表达,获得含有重组hGH蛋白的发酵液;
2)发酵液的处理:将步骤1)所得的发酵液5000 rpm离心5~10 min,取上清,过2 um滤膜,收集滤液;
3)超滤:将步骤2)得到的滤液用300kD超滤膜超滤,取截留液;
4)将步骤3)获得的截留液过Q离子交换柱,得到纯化的重组hGH蛋白。
4.如权利要求2所述的酵母重组子pPICZαA-hGH-GS115,其特征在于通过以下步骤获得:将pPICZαA-hGH载体线性化后,电转入GS115毕赤酵母菌株中,获得酵母重组子pPICZαA-hGH-GS115。
5.如权利要求3所述的重组hGH蛋白的纯化方法,其特征在于所述步骤1)当发酵培养至酵母湿重达到150 mg/ml以上时,加入适量甲醇,诱导 36-60h。
6.如权利要求3所述的重组hGH蛋白的纯化方法,其特征在于所述酵母重组子pPICZαA-hGH-GS115发酵培养条件为:温度30 ℃,pH 值为5.0,溶氧控制在25%~45%;甲醇诱导条件为:温度28 ℃,pH 值为5.6,溶氧控制在25%~45%。
7.如权利要求3所述的重组hGH蛋白的纯化方法,其特征在于步骤3)所述超滤为切向超滤。
8.如权利要求3所述的重组hGH蛋白的纯化方法,其特征在于步骤3)所述超滤包含以下步骤:将步骤2)得到的滤液调pH至6.9~7.5过300kD切向超滤膜超滤,压力0.1~0.2兆帕,当截留液为原体积的1/8~1/15时,加入5~10倍体积的pH7.2~7.5的10mmol/L 磷酸盐缓冲液,继续超滤至原体积1/8~1/15;重复3-8次。
9.如权利要求1所述的重组hGH蛋白的纯化方法,其特征在于步骤4)具体包含以下步骤:pH7.2~7.5的10 mmol/L 磷酸盐缓冲液即洗涤液平衡2~3个柱体积,上样,孵育3-5min,2~5倍柱体积洗涤液洗涤,含400 mmol/L NaCl的洗涤液洗脱,收集洗脱液, 脱盐,浓缩、干燥获得hGH重组蛋白。
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