CN108752459A - 半滑舌鳎igf-i蛋白及其体外表达制备方法与应用 - Google Patents
半滑舌鳎igf-i蛋白及其体外表达制备方法与应用 Download PDFInfo
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- CN108752459A CN108752459A CN201810553738.9A CN201810553738A CN108752459A CN 108752459 A CN108752459 A CN 108752459A CN 201810553738 A CN201810553738 A CN 201810553738A CN 108752459 A CN108752459 A CN 108752459A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
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- Health & Medical Sciences (AREA)
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- Chemical & Material Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Physics & Mathematics (AREA)
- Diabetes (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种半滑舌鳎IGF‑I蛋白及其体外表达制备方法与应用,所述的蛋白的核苷酸序列为SEQ ID NO:2;氨基酸序列为SEQ ID NO:3。本发明还提供一种半滑舌鳎IGF‑I蛋白的体外表达制备方法,它包括成熟肽序列改造、重组表达载体构建、重组表达载体诱导表达和重组蛋白纯化;本发明利用按照酵母密码子偏好性改造过的半滑舌鳎IGF‑I成熟肽序列与真核表达载体构建了重组表达载体,并在酵母工程菌中成功实现了IGF‑I的高效表达,获得了具有生物活性的半滑舌鳎IGF‑I重组蛋白,以饲料添加剂的形式应用后具有明显的促生长效果。
Description
技术领域
本发明属于生物基因工程技术领域,具体涉及一种半滑舌鳎IGF-I蛋白及其体外表达制备方法与应用。
背景技术
生长激素-***轴(GH/IGF-I axis)在鱼类生长发育过程中起着关键的生理调控作用。IGF-I是生长轴的下游调控因子,在进化上较为保守,其在硬骨鱼类生长、生殖、代谢、渗透压调节、行为、摄食和免疫等方面都具有重要的生理功能。为了促进IGF-I在鱼类养殖生产中应用,科研和养殖从业者希望能将IGF-I成熟肽在体外表达生产,获得IGF-I的蛋白产品后作为功能性生长代谢或免疫调节剂,以饲料添加剂等方式用于专用配合饲料研制。已有研究表明,重组IGF-I蛋白作为促生长或代谢调控因子,通过注射、浸泡或饲料添加的方式可有效促进养殖鱼类的生长代谢,且鱼类对外源重组蛋白尤其是同源的重组蛋白吸收快、代谢快,不会在体内产生累计,因而安全可靠。所以,利用基因工程表达手段获得重组鱼类类胰岛素生长因子并用于养殖鱼类生长调控,在水产养殖业中具有重要应用价值。目前,已利用基因工程手段在原核表达***(大肠杆菌)中成功实现了鲈鱼、罗非鱼、草鱼、虹鳟、大菱鲆等鱼类的IGF-I体外重组表达,获得了有生物活性的IGF-I重组蛋白,为鱼类IGF-I在养殖生产中的应用奠定了基础。但是,原核表达***存在许多难以克服的缺点:如原核宿主菌本身可能会产生内毒素,过量表达可能会导致非生理反应;原核表达***产生的目的蛋白常以包涵体形式表达,导致产物纯化困难;原核表达***翻译后加工修饰体系不完善,表达产物的生物活性较低。为克服原核表达载体的不足,研究者们开发了真核表达***,其优点是包括靶DNA与真核基因调控序列基本无同源性,避免了基因的非特异性激活或抑制;与原核表达相比,诱导基因表达效率显著提高;严格调控基因表达,获得的蛋白产物具有生物活性,无需分离、变性、复性等复杂处理过程。目前,基因工程研究中常用的真核表达***有酵母表达***、昆虫细胞表达***和哺乳动物细胞表达***。半滑舌鳎是我国重要的海水养殖经济鱼种,已成为我国鲆鲽鱼类养殖产业三大主导种类之一。半滑舌鳎具有明显的生长差异性别二态性,即雄鱼生长速度较雌鱼慢2-3倍,不利于产业的持续发展。如能利用具备生长代谢调控功能的基因产物来研制相应的生物制品,实现对养殖半滑舌鳎生长差异和代谢的人工调控,将会为养殖产业发展带来巨大经济效益,应用前景广阔。目前,已证明IGF-I在半滑舌鳎生长发育和代谢过程中具有重要的生理调控作用,但由于尚无成熟的IGF-I蛋白产品,因而尚未在养殖生产中应用。另外,目前市场上也未见半滑舌鳎养殖专用的功能性的生长代谢调控专用添加剂。
发明内容
为了解决上述技术难题,促进IGF-I在半滑舌鳎养殖生产中的应用,本发明提供一种半滑舌鳎IGF-I蛋白的体外真核表达制备及应用方法,利用按照酵母密码子偏好性改造过的半滑舌鳎IGF-I成熟肽序列,添加酶切位点和6×His标签序列后与真核表达载体构建了重组表达载体,并在酵母工程菌中成功实现了IGF-I的高效表达,获得了具有生物活性的半滑舌鳎IGF-I重组蛋白,以饲料添加剂的形式应用后具有明显的促生长效果。
本发明通过以下技术方案实现:
一种半滑舌鳎IGF-I蛋白,所述的蛋白的核苷酸序列为SEQ IDNO:2;
一种半滑舌鳎IGF-I蛋白,所述的蛋白的氨基酸序列为SEQ IDNO:3;
本发明提供一种半滑舌鳎IGF-I蛋白的体外表达制备方法,它包括成熟肽序列改造、重组表达载体构建、重组表达载体诱导表达和重组蛋白纯化;
所述的成熟肽序列改造,为了进行高效表达,对半滑舌鳎IGF-I成熟肽的核苷酸序列SEQ IDNO:1根据酵母菌的密码子偏好性进行改造,获得适宜在酵母菌内高效表达的核苷酸序列SEQ IDNO:2;
所述的重组表达载体构建,在目的核苷酸序列SEQ IDNO:2上添加酶切位点和His×6标签后与真核表达载体pPICZaA进行重组,构成可表达氨基酸序列为SEQ IDNO:3的半滑舌鳎类胰岛素生长因子I(IGF-I)多肽的重组表达载体pPICZaA-IGF-I。
进一步,添加酶切位点是指在目的核苷酸序列上游添加XhoI酶切位点,在下游添加XbaI酶切位点,并在目的核苷酸序列上游引入毕赤酵母蛋白酶Ste13酶切位点;
进一步,添加His×6标签是指添加于目的核苷酸序列下游,便于表达后目的蛋白的镍柱纯化。
所述的重组表达载体诱导表达,将重组表达载体线性化,电转导入处于化学感受态的表达菌株Pichia pastoris X33,形成重组表达菌株pPICZaA-IGF-I-Pichia pastorisX33,挑选高效表达的阳性克隆进行培养,并利用甲醇诱导重组表达菌分泌表达目的蛋白。
进一步,所述的重组表达载体诱导表达具体方法为取线性化的重组表达载体pPICZaA-IGF-I加入处于化学感受态的表达菌株Pichia pastoris X33,置于电转杯内,冰上放置6分钟后,利用电转仪转化,同时加入山梨醇,将电转后的液体转入离心管中30℃静止1.5h,然后转入YPD培养基中培育2h,培育条件:30℃,200rpm摇动,然后,涂布YPD平板,培养;
挑选平板培养的阳性克隆,以BMGY培养基培养,30℃200rpm,然后收集菌体利用BMMY培养至OD值至1.0时,加入甲醇诱导重组表达菌株分泌表达目的蛋白。
进一步,所述甲醇诱导浓度为0.5%;
进一步,所述诱导的条件为:温度为30℃,pH为5.0,诱导时间为36h。
所述的重组蛋白纯化,是指将培养的半滑舌鳎IGF-I重组表达酵母菌上清液以硫酸铵沉淀后再溶解,上清液经过微滤膜过滤后经过滤柱挂柱,再以洗脱液洗脱后即可得到纯化的目的蛋白。纯化蛋白的目的主要是测算目的蛋白产率和生物活性测定等科学研究,在生产应用过程中可不进行纯化而将酵母表达菌液直接拌入饲料中投喂应用。
进一步,硫酸铵浓度为350mg/L;
进一步,微滤膜孔径为0.45μm。
本发明与现有技术相比的有益效果:
(1)本发明使用的半滑舌鳎IGF-I成熟肽的核苷酸序列,根据表达酵母宿主菌的密码子偏好性改造后,与真核表达载体重组后的质粒导入Pichia pastoris X33酵母菌后表达效率提高2倍以上。
(2)本发明使用的真核表达载体pPICZaA属于分泌型表达载体,目的蛋白直接分泌于胞外,便于分离纯化,是目前应用最为广泛的一种真核表达载体。同时,pPICZaA表达载体的遗传背景清晰,具备完善的表达控制***和天然的翻译后修饰***,目的蛋白表达效率更高也更接近于天然蛋白质,具有稳定的自然生物活性,大大节省了生产工艺和人力物力。
(3)本发明将半滑舌鳎IGF-I真核重组表达载体在酵母中进行表达,可以借助酵母快速繁殖而实现重组蛋白的胞外量化分泌生产,同时,在获得了最佳诱导条件的基础上,还可利用发酵罐等工业化生产设备进行规模化培养,从而获得高产量而大大降低生产成本,具有较高经济性。酵母广泛应用于饲料与食品行业,本发明获得的IGF-I重组蛋白与酵母表达菌混合液可经真空干燥等处理后,作为添加剂直接应用于水产饲料,具有绿色、安全、生态、环保的优点。
附图说明
图1半滑舌鳎IGF-I重组蛋白在不同pH条件下的表达:1、pH3.0;2、pH4.0;3、pH5.0;4、pH6.0;5、pH7.0;
图2半滑舌鳎未改造(SEQ IDNO:1)和改造后(SEQ IDNO:2)IGF-I序列表达的重组蛋白:1、空白对照;2、SEQ IDNO:1序列表达的重组IGF-I蛋白;3、SEQ IDNO:2序列表达的重组IGF-I蛋白;
图3半滑舌鳎IGF-I重组蛋白对人乳腺癌细胞增殖的影响;
图4半滑舌鳎IGF-I重组蛋白对肝脏IGF-I mRNA和IGF-II mRNA表达的影响。
具体实施方式
下面通过实施例结合附图来对本发明的技术方案做进一步解释,但本发明的保护范围不受实施例任何形式上的限制。
实施例
一种半滑舌鳎IGF-I蛋白,所述的蛋白的核苷酸序列为SEQ IDNO:2;
所述的蛋白的氨基酸序列为SEQ IDNO:3;其体外表达制备方法,它包括成熟肽序列改造、重组表达载体构建、重组表达载体诱导表达和重组蛋白纯化。具体步骤如下:
1、成熟肽序列改造与合成
通过序列比对分析,获得了半滑舌鳎IGF-I基因成熟肽序列,全长204bp,由68个氨基酸组成,分子量约7.5kD,包括B、C、A、D 4个功能域。为了进行高效表达,对成熟肽进行核苷酸序列(SEQ IDNO:1)改造,通过人工合成的方式获得了适应酵母菌高效表达的IGF-I成熟肽的核苷酸序列片段SEQ IDNO:2,其氨基酸序列片段为SEQ IDNO:3。
为检测改造后成熟肽序列的表达效果,以SEQ IDNO:1序列为对照,同样以人工合成方式获得SEQ IDNO:1片段,同步开展重组蛋白表达试验。
2、重组表达载体构建
在目的核苷酸序列SEQ IDNO:2上游添加XhoI酶切位点,在下游添加XbaI酶切位点,同时在上游引入毕赤酵母蛋白酶Ste13酶切位点,并在序列下游His×6标签后与真核表达载体pPICZaA进行重组,构成可表达氨基酸序列为SEQ IDNO:3的半滑舌鳎类胰岛素生长因子I(IGF-I)多肽的重组表达载体pPICZaA-IGF-I。
对照试验组,在核苷酸序列SEQ IDNO:1上游添加XhoI酶切位点,在下游添加XbaI酶切位点,同时在上游引入毕赤酵母蛋白酶Ste13酶切位点,并在序列下游His×6标签后与真核表达载体pPICZaA进行重组,构成可表达氨基酸序列为SEQ IDNO:3的半滑舌鳎类胰岛素生长因子I(IGF-I)多肽的重组表达载体pPICZaA-IGF-I-C。以下试验操作步骤相同。
3、重组表达载体诱导表达
制备处于化学感受态的Pichia pastoris X33菌株。取10ml线性化的重组表达载体pPICZaA-IGF-I加入80ml处于化学感受态的表达菌株Pichia pastoris X33,置于电转杯内,冰上放置6分钟后,利用电转仪转化,同时加入1ml 1M山梨醇,将电转后的液体转入离心管中30℃静止1.5h,然后转入YPD培养基中培育2h,培育条件:30℃,200rpm摇动。然后,涂布YPD平板,培养。
挑选平板培养的阳性克隆,以BMGY培养基培养(30℃,200rpm),然后收集菌体利用BMMY培养至OD值至1.0时,加入甲醇诱导重组表达菌株分泌表达目的蛋白。试验确定最佳诱导条件为:甲醇浓度为0.5%,温度为30℃,pH为5.0,诱导时间为36h。
4、重组蛋白纯化
在甲醇诱导表达36h后,取培养的酵母表达菌液的上清液以350mg/L硫酸铵沉淀(4℃),再以洗脱缓冲液溶解后经过0.45μm微滤膜过滤。过滤液经过His标签吸附柱过滤挂柱,再以洗脱液将蛋白洗脱下来,即可得到纯化的目的蛋白。目的蛋白以Tricine–SDS–PAGE和western blot方法验证确认。目的蛋白以BCA法和Tricine–SDS–PAGE凝胶图像分析法获得最终浓度。
蛋白重组表达结果显示,经改造后的半滑舌鳎IGF-I成熟肽序列在毕赤酵母X33菌株中获得了高效表达(图1、图2),表达产量达3.95g/L,而未经改造的IGF-I多肽序列表达产量仅为1.14g/L(图2),表达效率提升2.46倍,可见改造后的半滑舌鳎IGF-I成熟肽序列较未经改造的IGF-I成熟肽序列在酵母中具有高效率的表达。
5、生物活性测定
以获得的纯化的半滑舌鳎IGF-I蛋白作用于人乳腺癌细胞和半滑舌鳎肝脏细胞,测试获得的重组目的蛋白是否具有生物活性。结果表明,获得的半滑舌鳎IGF-I重组蛋白可显著刺激人乳腺癌细胞增殖(图2),同时还可显著调控肝脏IGF-I、IGF-II等基因的表达(图3),表明获得的半滑舌鳎IGF-I重组蛋白具有明显的生物活性。
6、半滑舌鳎IGF-I重组蛋白的应用
本发明提供的半滑舌鳎IGF-I蛋白体外真核表达制备方法可成功实现半滑舌鳎IGF-I成熟肽在酵母中的高效表达,适宜发酵等工业化生产,可规模化制备。将分离纯化的半滑舌鳎IGF-I重组蛋白,以去离子水保存成水剂,可用于科学实验研究。例如,按照2.5μg/kg和25μg/kg的IGF-I重组蛋白剂量,分别腹腔注射半滑舌鳎幼鱼,每两周一次,在45d内体重增加较对照组分别提高35.75%和50.91%,促生长效果非常明显。在进行喷雾干燥、冻干等处理后可获得半滑舌鳎IGF-I重组蛋白的酵母粉剂产品,直接可应用于饲料添加剂使用。以0.1%的比例添加到饲料中,投喂半滑舌鳎幼鱼60d,投喂组实验鱼体重增长率较对照组实验鱼高30.7%,促生长效果明显。
序列表
<110> 中国水产科学研究院黄海水产研究所
<120> 半滑舌鳎IGF-I蛋白及其体外表达制备方法与应用
<130> wu
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 204
<212> DNA
<213> Cynoglossus semilaevis
<400> 1
ggcccggaga ccctgtgcgg ggcggagctg gtcgacacgc tgcagtttgt gtgtggagag 60
agaggctttt atttcagcaa accaacwggc tatggcccta actcacggcg gtctcgtggc 120
atcgtggacg agtgctgctt ccaaagctgt gagctgcggc gcctggagat gtactgcgcg 180
ccagccaaga ctggcaaagc agct 204
<210> 2
<211> 204
<212> DNA
<213> Cynoglossus semilaevis
<400> 2
ggtccagaga ctctgtgtgg tgctgagctg gttgacactc tgcaattcgt ttgtggtgag 60
agaggtttct acttcagtaa gccaactggt tacggtccaa actctcgtcg ttctcgtggt 120
attgttgacg agtgttgttt ccaaagttgt gagctgcgtc gtctggagat gtactgtgct 180
ccagccaaga ctggtaaggc tgct 204
<210> 3
<211> 68
<212> PRT
<213> Cynoglossus semilaevis
<400> 3
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Thr Leu Gln Phe
1 5 10 15
Val Cys Gly Glu Arg Gly Phe Tyr Phe Ser Lys Pro Thr Gly Tyr Gly
20 25 30
Pro Asn Ser Arg Arg Ser Arg Gly Ile Val Asp Glu Cys Cys Phe Gln
35 40 45
Ser Cys Glu Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Ala Lys Thr
50 55 60
Gly Lys Ala Ala
65
Claims (10)
1.一种半滑舌鳎IGF-I蛋白,其特征在于所述半滑舌鳎IGF-I蛋白的核苷酸序列为SEQIDNO:2。
2.权利要求1所述的一种半滑舌鳎IGF-I蛋白,其特征在于所述半滑舌鳎IGF-I蛋白的氨基酸序列为SEQ IDNO:3。
3.权利要求1所述半滑舌鳎IGF-I蛋白的体外表达制备方法,其特征在于它包括成熟肽序列改造、重组表达载体构建、重组表达载体诱导表达和重组蛋白纯化;
所述的成熟肽序列改造,对半滑舌鳎IGF-I成熟肽的核苷酸序列SEQ IDNO:1根据酵母菌的密码子偏好性进行改造,获得适宜在酵母菌内高效表达的核苷酸序列SEQ IDNO:2;
所述的重组表达载体构建,在目的核苷酸序列SEQ IDNO:2上添加酶切位点和His×6标签后与真核表达载体pPICZaA进行重组,构成可表达氨基酸序列为SEQ IDNO:3的半滑舌鳎类胰岛素生长因子I多肽的重组表达载体pPICZaA-IGF-I;
所述的重组表达载体诱导表达,将重组表达载体线性化,电转导入处于化学感受态的表达菌株Pichia pastoris X33,形成重组表达菌株pPICZaA-IGF-I-Pichia pastorisX33,挑选高效表达的阳性克隆进行培养,并利用甲醇诱导重组表达菌分泌表达目的蛋白。
4.根据权利要求3所述的方法,其特征在于所述的重组表达载体构建步骤中所述添加酶切位点是指在目的核苷酸序列上游添加XhoI酶切位点,在下游添加XbaI酶切位点,并在目的核苷酸序列上游引入毕赤酵母蛋白酶Ste13酶切位点。
5.根据权利要求3所述的方法,其特征在于所述的重组表达载体构建步骤中添加His×6标签是指添加于目的核苷酸序列下游。
6.根据权利要求3所述的方法,其特征在于所述的重组表达载体诱导表达具体方法为取线性化的重组表达载体pPICZaA-IGF-I加入处于化学感受态的表达菌株Pichiapastoris X33,置于电转杯内,冰上放置6分钟后,利用电转仪转化,同时加入山梨醇,将电转后的液体转入离心管中30℃静止1.5h,然后转入YPD培养基中培育2h,培育条件:30℃,200rpm摇动,然后,涂布YPD平板,培养;
挑选平板培养的阳性克隆,以BMGY培养基培养,30℃200rpm,然后收集菌体利用BMMY培养至OD值至1.0时,加入甲醇诱导重组表达菌株分泌表达目的蛋白。
7.根据权利要求6所述的方法,其特征在于所述甲醇诱导浓度为0.5%。
8.根据权利要求6所述的方法,其特征在于所述诱导的条件为:温度为30℃,pH为5.0,诱导时间为36h。
9.根据权利要求3所述的方法,其特征在于所述的重组蛋白纯化,是指将培养的半滑舌鳎IGF-I重组表达酵母菌上清液以硫酸铵沉淀后再溶解,上清液经过微滤膜过滤后经过滤柱挂柱,再以洗脱液洗脱后即可得到纯化的目的蛋白。
10.根据权利要求9所述的方法,其特征在于所述的硫酸铵浓度为350mg/L、微滤膜孔径为0.45μm。
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