WO2022022197A1 - 一种基于n端编码序列改造调控蛋白质表达的方法 - Google Patents
一种基于n端编码序列改造调控蛋白质表达的方法 Download PDFInfo
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- the invention relates to a method for regulating protein expression based on the modification of an N-terminal coding sequence, belonging to the technical field of genetic engineering and enzyme engineering
- Mutation of genes is of great significance for changing the properties of proteins. Usually, mutation sequences with better properties can be found through mutation, thereby improving the application value of proteins. Synonymous mutation of genes is a commonly used mutation method, and the expression levels of synonymous mutations of genes can vary greatly.
- the current commonly used method is to construct a synonymous mutation library and combine it with a high-throughput screening strategy to find the best mutant.
- this method is time-consuming, labor-intensive, and specific, and cannot be used to guide the design of other genes.
- synthesizing a series of short peptides is beneficial to widely improve gene expression, this method will have an impact on enzyme activity, because these expression-promoting short peptides occupy the position of the signal peptide, so that the Suitable for extracellular proteins that require the addition of a signal peptide.
- NCS N-terminal coding region
- the method of the present invention is established based on the bioinformatics analysis of representative samples, and by this method, the nucleotide sequence of the first 30 bases of the N-terminal of any gene can be de novo designed, and synonymous mutation can be performed on it.
- the NCS nucleotide sequence of any gene is changed to the target nucleotide sequence by mutating the primers.
- the present invention can be used to guide the design of any gene without adding additional amino acid sequence, and the properties of the protein are minimized. It can greatly improve the expression level of the target gene.
- the present invention provides a method for screening nucleotide sequences encoding proteins with different expression levels, measuring the values of GC3 and ⁇ G, and then calculating the relative expression level of the protein by using the following equation, that is, the PsfGFP value, and screening according to the PsfGFP value The corresponding nucleotide sequence is obtained; the P sfGFP value is positively correlated with the actual expression of the protein:
- PsfGFP 274497.657-108717.401 ⁇ GC3+4886.529 ⁇ G.
- the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.
- the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 96 bp region of the N-terminal coding region.
- the protein is any protein that can be expressed in Bacillus subtilis.
- the protein includes, but is not limited to, pullulanase.
- amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
- the PsfGFP value is positively correlated with the actual expression level of the protein.
- the corresponding nucleotide sequences are screened according to the PsfGFP value.
- the invention provides a method for regulating the protein expression of genetically engineered bacteria.
- GC3 and ⁇ G parameters of the gene calculate the relative expression level of each nucleotide sequence according to the equation, select the nucleotide sequence with the required expression level, mutate the N-terminal coding region of the target protein accordingly, and transform it into in host cells;
- the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.
- the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 96 bp region of the N-terminal coding region.
- the nucleotide sequences whose P sfGFP value in the mutation library is in the top 10% are selected; when the protein expression needs to be down-regulated, the P sfGFP value in the mutation library is selected. sfGFP values are in the bottom 10% of nucleotide sequences.
- the genetically engineered bacteria use Bacillus subtilis as a host.
- the protein is any protein that can be expressed in Bacillus subtilis.
- the protein includes, but is not limited to, pullulanase.
- amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
- the invention provides a method for regulating the expression level of pullulanase.
- the ⁇ G is the minimum free energy of the mRNA secondary structure between the transcription initiation site of any promoter of the gene encoding the target protein and the 90-99 bp region of the N-terminal coding region.
- the recombinant plasmid is introduced into Bacillus subtilis, and the Bacillus subtilis is used to produce the protein.
- amino acid sequence of the pullulanase is shown in SEQ ID NO.19.
- the present invention also protects the application of the method for screening nucleotide sequences encoding high-expression proteins, or the method for regulating the protein expression of genetically engineered bacteria in regulating the expression of a target protein.
- the present invention also protects the application of the method for regulating the expression of pullulanase in regulating pullulanase.
- the calculated PsfGFP value is positively correlated with the actual expression level of the protein.
- to calculate the PsfGFP value is to select the corresponding synonymous mutation sequence as needed. It was applied to transform the N-terminus of pullulanase fused to the nucleotide sequence of the Bgls signal peptide, and the selected synonymous mutation sequence could increase the extracellular enzyme activity by 2.67 times and decrease it by 48%.
- Figure 1 is the map of the sfGFP expression plasmid P43-NMK-sfGFP.
- Figure 2 is a graph showing the relative fluorescence intensity of the NCS library of sfGFP.
- Figure 3 shows the nucleotide sequence indices and fluorescence values of the 1st to 60th samples among the 172 samples.
- Figure 4 shows the nucleotide sequence indices and fluorescence values of the 61st to 120th samples among the 172 samples.
- Figure 5 shows the nucleotide sequence indexes and fluorescence values of the 121st to 172nd samples in the 172 samples.
- Figure 6 is the distribution of relative fluorescence values before and after transformation.
- Figure 7 is the map of pullulanase expression plasmid P43-NMK-Bgls fused with BglS signal peptide.
- Figure 8 is a protein gel image of the 5 NCS variants of the BglS signal peptide.
- Figure 9 is a graph showing the correlation between the predicted expression value of pullulanase with the addition of five Bgls signal peptide sequences and the measured value of enzyme activity.
- Seed medium (g/L): peptone 10, yeast extract 5, sodium chloride 5;
- Fermentation medium (g/L): The following components were dissolved in 0.9L water: peptone 12g, yeast extract 24g, glycerol 4mL.
- Seed culture Pick a single colony of engineering bacteria and insert it into the seed medium, the culture temperature is 37°C, the shaking speed is 200r/min, and the culture is 24h;
- Fermentation culture The seed culture liquid is inserted into the fermentation medium according to the inoculum amount of 4%, the culture temperature is 37 °C, and the fermentation is carried out for 24 hours.
- One-step cloning kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.
- Glue concentration of 10% SDS-PAGE gel was used to analyze the protein expression level.
- MES or MOPS buffer was used as the running buffer, and the loading volume was 10 ⁇ L.
- the electrophoresis voltage was 150V.
- Specific sample preparation and electrophoresis operations were performed according to the kit instructions.
- the molecular weights (kDa) of the standard protein were: 188, 98, 62, 49, 38, 28, 17, 14, 6 and 3; and when electrophoresed in MOPS buffer, the molecular weight of the standard protein was Molecular weights (kDa) are: 191, 97, 64, 51, 39, 28, 19, 14
- the PLytr promoter (nucleotide sequence shown in SEQ ID NO.1) was used with primers Lytr-F/Lytr-R (nucleotide sequence shown in SEQ ID NO.2 and 3) and Lytr-F-plasmid/ Lytr-R-plasmid (nucleotide sequence shown in SEQ ID NO. 4 and 5) was connected to the P43NMK plasmid by a one-step cloning kit to construct the plasmid P43NMK-Lytr;
- the sfGFP fluorescent protein reporter gene (nucleotide sequence shown in SEQ ID NO.6) was used primers sfGFP-F/sfGFP-R (nucleotide sequence shown in SEQ ID NO.7 and 8) and sfGFP-F-plasmid/sfGFP-R-plasmid (nucleotide sequences shown in SEQ ID NO. 9 and 10) were fused to the downstream of PLytr by a one-step cloning kit to obtain the construction of P43NMK-Lytr_sfGFP, as shown in Figure 1. Show;
- the recombinant plasmids with synonymous mutations constructed in Example 1 were transformed into the expression host Bacillus subtilis WB600, respectively, and the transformed single clones were inoculated into 96 shallow-well plates containing 200 ⁇ L of LB seed medium, and cultured for 8 hours;
- a total of 8598 monoclonal host cells were characterized in Example 2, and the fluorescence value/OD was defined as the relative fluorescence intensity RFI. According to the level of the RFI value, the monoclonal cells were sorted from high to low, and every 50 cells were selected for sequencing identification (that is, the first One of the 1 to 50 strains was selected, one of the 51 to 100 strains was selected, and so on), and a total of 172 single clones were identified by sequencing.
- GC3 The third base of the synonymous codon is the content of GC;
- T3s, C3s, A3s, G3s After a synonymous mutation occurs at the first 30 bases of the N-terminal of the gene, the third synonymous codon is the frequency of T, C, A, and G, respectively;
- CAI codon preference
- Fop frequency of optimal codons (both above calculated ranges are 30 nucleotide sequences for NCS mutations).
- ⁇ G the minimum free energy
- the calculated range includes the region from the transcription start site to the downstream of NCS, in this example, 25 bases upstream of ATG (the transcription start site of the PLytr promoter) to 96 bases downstream of ATG were selected base;
- TIR translation initiation rate
- the range is the same as the calculation ⁇ G.
- Example 3 Substitute the sequence of 172 samples in Example 3 into the regression prediction equation, calculate the predicted value, and compare it with the actual fluorescence value measured in Example 3, and perform correlation analysis. As shown in Figure 6, the sequence of The Pearson coefficient between the predicted value and the measured fluorescence value can reach 0.675, and the correlation is very strong, indicating that the regression prediction equation can be used to predict the protein fluorescence value.
- Example 5 Using a prediction equation to guide NCS engineering of the signal peptide BglS gene
- the BglS signal peptide (nucleotide sequence shown in SEQ ID NO. 13) was fused to the N-terminal of the pullulanase encoding gene (nucleotide sequence shown in SEQ ID NO. 14) to achieve pullulanase of extracellular expression.
- the specific method is to clone the BglS signal peptide into the downstream of PLytr in P43NMK-Lytr by using the same one-step cloning method in the example to construct P43NMK-Lytr-BglS, as shown in FIG. 7 .
- NCS region of BglS close to ATG was optimized: all the synonymous mutation combinations of the first ten amino acids of BglS were exhausted, and there were 131,072 possibilities; according to the examples 4 equations were used to calculate the GC3 and ⁇ G of each of the 131072 sequences and the theoretical value of PsfGFP, and according to the predicted value, 5 Bgls variants including wild type were selected: NCS+, NCS+', NCS-wt, NCS -', NCS-.
- NCS+ represents the P sfGFP maximum variant
- NCS+' represents the intermediate variant between the maximum and wild type of P sfGFP
- NCS-wt represents the wild type
- NCS- represents the P sfGFP minimum variant
- NCS-' represents the intermediate variant Between the minimum of PsfGFP and the intermediate value variant of wild type, it has continuously decreasing predicted expression intensity.
- step (1) signal peptide Bgls variants NCS+ (nucleotide sequence shown in SEQ ID NO. 15), NCS+' (nucleotide sequence shown in SEQ ID NO. 16), NCS- ' (the nucleotide sequence is shown in SEQ ID NO. 17), NCS- (the nucleotide sequence is shown in SEQ ID NO.
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Abstract
本发明公开了一种基于N端编码序列改造调控蛋白质表达的方法,这属于基因工程领域。本发明是以枯草芽孢杆菌为表达宿主,通过预测模型,评价N端编码区同义突变中,最有利于促进基因表达的核苷酸序列。通过结合超折叠绿色荧光蛋白(sfGFP)的NCS的前十个氨基酸同义突变文库,测定文库中蛋白的荧光强度,选择172个代表性样本并测序鉴定,使用统计学方法建立预测模型。通过该模型优化融合了BlgS信号肽的普鲁兰酶,可使普鲁兰酶胞外酶活提高至改造前的2.67倍以及降低48%,从而为N端基因的从头设计提供理性改造的方向,有利于简易地调控基因的表达。
Description
本发明涉及一种基于N端编码序列改造调控蛋白质表达的方法,属于基因工程及酶工程技术领域
基因的突变对于改变蛋白的性质具有非常重要的意义,通常通过突变,可以从中找到性质更好的突变序列,从而提高蛋白的应用价值。基因的同义突变就是常用的一种突变手段,基因的同义突变可实现表达量相差巨大。
目前常用的方法:是通过构建同义突变文库,并结合高通量筛选策略,以期找到最佳突变体。然而这种方法耗时耗力,并且专一性强,无法用于指导其他基因的设计。尽管有的研究通过发现,合成一系列的短肽,有利于广泛提高基因的表达,然而这种方法会对酶活产生影响,由于这些促表达的短肽,占据了信号肽的位置,从而不适合于需要添加信号肽的胞外蛋白。
现有用于改善基因表达量的方法,往往都是通过非翻译区(5’UTR)的优化,然而当5’UTR模块已经足够强时,难以继续优化并显著提高表达量。而关于N端编码区(NCS)的研究较少。因此,建立一种适用于广泛基因设计的NCS改造策略非常重要。
发明内容
本发明的方法是基于对代表性样本的生物信息学分析而建立的,通过此方法,可从头设计任意基因的N端前30位碱基的核苷酸序列,对其进行同义突变。本模型的实施方案中,是通过突变引物,改变任意基因的NCS核苷酸序列为目的核苷酸序列所完成。本发明通过优化NCS的核苷酸序列,可用于指导任意基因的设计,并不需要添加额外的氨基酸序列,对蛋白质的性质降到最低。可极大的提高目的基因的表达水平。
本发明提供了一种筛选编码不同表达量的蛋白的核苷酸序列的方法,测定GC3和ΔG的值,再应用下述方程式计算蛋白的相对表达量,即P
sfGFP值,根据P
sfGFP值筛选出对应的核苷酸序列;P
sfGFP值与蛋白的实际表达量成正相关:
P
sfGFP=274497.657-108717.401×GC3+4886.529×ΔG。
在一种实施方式中,所述GC3为编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸的同义密码子第三位碱基是GC的含量,n=3;所述ΔG为编码目的蛋白的基因的任意启动子转录起始位点至N端编码区的第90~99bp区域间的mRNA二级结构的最小自由能。
在一种实施方式中,所述ΔG为编码目的蛋白的基因的任意启动子转录起始位点至N端编码区的第96bp区域间的mRNA二级结构的最小自由能。
在一种实施方式中,所述蛋白为能够在枯草芽孢杆菌中表达的任意蛋白。
在一种实施方式中,所述蛋白包括但不限于普鲁兰酶。
在一种实施方式中,所述普鲁兰酶的氨基酸序列如SEQ ID NO.19所示。
在一种实施方式中,P
sfGFP值与蛋白的实际表达量呈正相关。
在一种实施方式中,根据P
sfGFP值筛选相应的核苷酸序列。
在一种实施方式中,P
sfGFP值越高,对应的蛋白表达量越高。
本发明提供了一种调控基因工程菌蛋白表达量的方法,选取目的蛋白N端编码区的长度为9n~10n个核苷酸,n=3,建立同义突变库;计算同义突变库中的基因的GC3和ΔG参数,根据方程计算每个核苷酸序列的相对表达量,选择具有所需表达量的核苷酸序列,将目的蛋白N端编码区进行相应突变,并将其转化到宿主细胞中;
所述方程为:P
sfGFP=274497.657-108717.401×GC3+4886.529×ΔG。
在一种实施方式中,所述GC3为编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸的同义密码子第三位碱基是GC的含量,n=3。
在一种实施方式中,所述ΔG为编码目的蛋白的基因的任意启动子转录起始位点至N端编码区的第90~99bp区域间的mRNA二级结构的最小自由能。
在一种实施方式中,所述ΔG为编码目的蛋白的基因的任意启动子转录起始位点至N端编码区的第96bp区域间的mRNA二级结构的最小自由能。
在本发明的一种实施方式中,蛋白表达量需要上调的时候,选择突变库中的P
sfGFP值处于前10%的核苷酸序列;蛋白表达量需要下调的时候,选择突变库中的P
sfGFP值处于后10%的核苷酸序列。
在一种实施方式中,将编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸突变为相应的核苷酸序列,n=3。
在一种实施方式中,所述基因工程菌以枯草芽孢杆菌为宿主。
在一种实施方式中,所述蛋白为能够在枯草芽孢杆菌中表达的任意蛋白。
在一种实施方式中,所述蛋白包括但不限于普鲁兰酶。
在一种实施方式中,所述普鲁兰酶的氨基酸序列如SEQ ID NO.19所示。
本发明提供了一种调控普鲁兰酶表达量的方法,将选取普鲁兰酶N端编码区前9n~10n个核苷酸,n=3,进行同义突变,构建突变体库,并计算P
sfGFP值,根据P
sfGFP值选择相应的 同义突变序列;将目的蛋白的N端编码区进行相应突变,连接至表达载体,构建重组质粒;
所述P
sfGFP值按照下述方式计算:P
sfGFP=274497.657-108717.401×GC3+4886.529×ΔG;
所述GC3为编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸的同义密码子第三位碱基是GC的含量,n=3;
所述ΔG为编码目的蛋白的基因的任意启动子转录起始位点至N端编码区的第90~99bp区域间的mRNA二级结构的最小自由能。
在一种实施方式中,普鲁兰酶表达量需要上调的时候,选择突变库中的P
sfGFP值处于前10%的核苷酸序列,将编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸突变为相应的核苷酸序列,n=3。
在一种实施方式中,普鲁兰酶表达量需要下调的时候,选择突变库中的P
sfGFP值处于后10%的核苷酸序列,将编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸突变为相应的核苷酸序列,n=3。
在一种实施方式中,将重组质粒导入枯草芽孢杆菌,利用枯草芽孢杆菌生产蛋白。
在一种实施方式中,所述普鲁兰酶的氨基酸序列如SEQ ID NO.19所示。
本发明还保护所述筛选编码高表达量蛋白的核苷酸序列的方法,或调控基因工程菌蛋白表达量的方法在调节目的蛋白表达量中的应用。
本发明还保护所述调控普鲁兰酶表达量的方法在调节普鲁兰酶中的应用。
本发明的有益效果:
本发明通过结合sfGFP、并对目的基因的N端编码区进行改造(同义突变),探究出了一条用于指导蛋白做出定向改造、从而提高或降低目的蛋白表达的公式PsfGFP=274497.657-108717.401×GC3+4886.529×ΔG。所算得的PsfGFP值与蛋白的实际表达量成正相关,根据此公式,计算出PsfGFP值即根据需要选择相应的同义突变序列。将其应用于改造融合了Bgls信号肽的核苷酸序列的普鲁兰酶N端,选择的同义突变序列可使胞外酶活上调2.67倍、以及下调48%。
图1为sfGFP表达质粒P43-NMK-sfGFP图谱。
图2为sfGFP的NCS文库相对荧光强度情况图。
图3为172个样本中第1~60个样本的核苷酸序列指标和荧光值。
图4为172个样本中第61~120个样本的核苷酸序列指标和荧光值。
图5为172个样本中第121~172个样本的核苷酸序列指标和荧光值。
图6为改造前后相对荧光值分布图。
图7为融合BglS信号肽的普鲁兰酶表达质粒P43-NMK-Bgls图谱。
图8为BglS信号肽的5种NCS变体蛋白胶图。
图9为添加5种Bgls信号肽序列的普鲁兰酶的表达预测值与酶活测量值相关性图。
1、培养基组成:
种子培养基(g/L):蛋白胨10,酵母提取物5,氯化钠5;
发酵培养基(g/L):将下列组分溶解在0.9L水中:蛋白胨12g,酵母提取物24g,甘油4mL。
各组分溶解后高压灭菌;冷却到60℃,再加100mL灭菌的0.17mol/L的KH2PO4、0.72mol/L的K2HPO4溶液(2.31g的KH2PO4和12.54g的K2HPO4溶在足量的水中,使终体积为100mL;0.22μm的滤膜过滤除菌);
2、培养方法:
种子培养:挑取工程菌单菌落接入种子培养基中,培养温度37℃,摇床转速200r/min,培养24h;
发酵培养:种子培养液按4%的接种量接入发酵培养基中,培养温度37℃,发酵24h
3、绿色荧光蛋白表达量及生物量测定
在96孔板中加入用PBS缓冲液(100mM和pH 7.2)稀释成合适浓度的发酵液,使用Cytation3细胞成像微孔板检测仪(美国伯腾仪器有限公司),绿色荧光激发波长:480nm,绿色荧光发射波长:520nm,细胞生长OD吸收波长:600nm。
一步克隆试剂盒购自南京诺唯赞生物科技有限公司。
4、SDS-PAGE电泳检测
胶浓度为10%的
SDS-PAGE胶被用于分析蛋白的表达水平,以MES或MOPS缓冲液为电泳缓冲液,上样量为10μL。电泳电压为150V。具体样品制备及电泳操作依照试剂盒说明书进行。以MES缓冲液进行电泳时,标准蛋白的分子量(kDa)分别为:188,98,62,49,38,28,17,14,6和3;而以MOPS缓冲液进行电泳时,标准蛋白的分子量(kDa)分别为:191,97,64,51,39,28,19,14
5、普鲁兰酶酶活测定方式
将1mL 1g/100mL普鲁兰多糖底物和0.9mL 100mM pH 4.5乙酸-乙酸钠缓冲液混合均匀,置于60℃水浴锅内预热10min,加入普鲁兰酶液0.1mL,反应10min后,加入3mL DNS显色液,然后于沸水浴中煮7min,置于冰水中终止显色反应,再加10mL去离子水, 混匀,在540nm下测定吸光值。单位时间内生成1μmol还原糖的酶量定义为一个酶活力单位。
实施例1:构建NCS同义突变文库
将PLytr启动子(核苷酸序列如SEQ ID NO.1所示)使用引物Lytr-F/Lytr-R(核苷酸序列如SEQ ID NO.2和3所示)和Lytr-F-plasmid/Lytr-R-plasmid(核苷酸序列如SEQ ID NO.4和5所示)通过一步克隆试剂盒连接至P43NMK质粒,构建得到质粒P43NMK-Lytr;
采用相同的手段,将sfGFP荧光蛋白报告基因(核苷酸序列如SEQ ID NO.6所示)使用引物sfGFP-F/sfGFP-R(核苷酸序列如SEQ ID NO.7和8所示)和sfGFP-F-plasmid/sfGFP-R-plasmid(核苷酸序列如SEQ ID NO.9和10所示),通过一步克隆试剂盒融合至PLytr的下游,得到构建P43NMK-Lytr_sfGFP,如图1所示;
以P43NMK-Lytr_sfGFP为模板,使用简并引物sfGFP-F-NCS/sfGFP-R-NCS(核苷酸序列如SEQ ID NO.11和12所示),获得sfGFP的N端前30位碱基发生同义突变的重组质粒,这些重组质粒构成了同义突变文库,使得sfGFP前30个碱基发生改变,但其编码的氨基酸序列保持不变。
实施例2:NCS同义突变文库的表征
将实施例1中构建得到的发生同义突变的重组质粒分别转化至表达宿主枯草芽孢杆菌WB600中,将转化后的单克隆接种至含有200μL LB种子培养基的96浅孔板,培养8小时;
接着,按照4mL/100mL的接种量接种至含有800μL TB培养基的96深孔板,培养24小时得到发酵液;
然后将发酵液迅速置于冰上冷冻,离心后,去除上清,用PBS缓冲液(100mM、pH 7.2)稀释至合适倍数后,通过Cytation3细胞成像微孔板检测仪(美国伯腾仪器有限公司)测定荧光值(激发光480,吸收光520)以及OD
600。共表征了8598个单菌落,如图2。
实施例3:代表性样本的序列鉴定和发酵
实施例2中共表征8598个单克隆宿主细胞,定义荧光值/OD为相对荧光强度RFI,根据RFI值的高低,将单克隆细胞由高到低排序,每50个选择1个测序鉴定(即第1~50个菌株中选择一个,第51~100个菌株中选择一个,依此类推),共测序鉴定了172个单克隆。
将172个经测序鉴定后的单克隆,接种至含有20mL种子培养基的250mL摇瓶中,37℃、220rpm发酵8小时后至OD
600大于4,按照4mL/100mL的比例接种到含有25mL发酵培养基的250mL摇瓶中,发酵24小时后,测定sfGFP的荧光值和OD
600。每组实验设置3个平行。其结果如下图3~5。
实施例4:使用生物信息学工具对样本的核苷酸进行序列分析
使用CodonW、Nupack、RBS calculator创建11个不同的核苷酸序列指标以进行序列分析。
(1)使用CodonW计算GC、GC3、T3s、C3s、A3s、G3s、CAI、CBI、Fop
GC:目的基因的G+C含量;
GC3:同义密码子第三位碱基是GC的含量;
T3s、C3s、A3s、G3s:基因的N端前30位碱基发生同义突变后,第三个同义位置密码子分别是T、C、A、G的频率;
CAI:密码子偏好性;
CBI:密码子偏爱指数;
Fop:最佳密码子的频率(上述计算范围均是NCS突变的30个核苷酸序列)。
(2)使用Nupack计算ΔG
ΔG:最小自由能,其计算的范围包含转录起始位点至NCS下游的区域,在本实施例中选取ATG上游25个碱基处(PLytr启动子的转录起始位点)至ATG下游96碱基处;
(3)使用RBS calculator计算TIR
TIR:翻译起始率,范围同计算ΔG。
通过对172个样本中,以RFI作为因变量,11个核苷酸序列指标作为因变量进行分析,通过SPSS进行多元回归分析,方法采用逐步回归。
最终获得一条回归预测方程PsfGFP=274497.657-108717.401×GC3+4886.529×ΔG,见表1。并用以指导基因的NCS改造,在对NCS进行改造时,通过计算相应参数带入公式,即能根据算出的值,选择蛋白表达量高的同义突变序列。
表1 多元回归分析
将实施例3中的172个样本的序列代入所述回归预测方程,计算出预测值,并与实施例3中测定的实际荧光值进行比较,进行相关性分析,如图6所示,序列的预测值和测量荧光值之间的皮尔逊系数可达0.675,相关非常强,说明所述的回归预测方程可以用来预测蛋白荧光值。
实施例5:使用预测方程指导信号肽BglS基因的NCS改造
(1)P43NMK-Lytr_BglS野生型的构建
将BglS信号肽(核苷酸序列如SEQ ID NO.13所示)融合在普鲁兰酶编码基因(核苷酸序列如SEQ ID NO.14所示)的N端,实现了普鲁兰酶的胞外表达。具体方式为利用实施例相同的一步克隆法,将BglS信号肽克隆至P43NMK-Lytr中的PLytr的下游,构建得到P43NMK-Lytr-BglS,如图7。
(2)P43NMK-Lytr_BglS同义突变质粒的构建
为了进一步的提高普鲁兰酶的胞外酶活,优化了靠近ATG的BglS的NCS区:将BglS的前十个氨基酸所有的同义突变组合方式穷举出来,共有131072种可能;按照实施例4的方程进行计算,计算131072条序列每一条序列的GC3和ΔG以及理论值PsfGFP,并根据预测值,选择包括野生型在内的5种Bgls变体:NCS+,NCS+’,NCS-wt,NCS-’,NCS-。
NCS+代表P
sfGFP最大值变体;NCS+’代表P
sfGFP的最大值与野生型之间的中间值变体;NCS-wt代表野生型;NCS-代表P
sfGFP最小值变体,NCS-’代表介于P
sfGFP的最小值与野生型之间的中间值变体,其具有连续降低的预测表达强度。
利用与步骤(1)相同的方法,信号肽Bgls变体NCS+(核苷酸序列如SEQ ID NO.15所示)、NCS+’(核苷酸序列如SEQ ID NO.16所示)、NCS-’(核苷酸序列如SEQ ID NO.17所示)、NCS-(核苷酸序列如SEQ ID NO.18所示),分别连接至克隆至P43NMK-Lytr中的PLytr的下游,分别得到含有BglS信号肽同义突变序列的质粒;再将得到的质粒转化至表达宿主枯草芽孢杆菌WB600中,将转化后的单克隆接种至含有20mL LB培养基的250mL摇瓶中,37℃ 220rpm发酵8小时后,使得OD
600达到4以上,按照4mL/100mL的比例接种到含有25mL TB培养基的250mL摇瓶中,在37℃、250rpm发酵30小时后,测定普鲁兰酶的胞外酶活,结果如图9所示,发现其普鲁兰酶胞外酶活实现了预测的高中低5水平变化,并且与预测值有0.89的R
2水平。
表2 信号肽BglS的NCS突变体的预测及实际检测结果
预测荧光值(×10 3) | 发酵酶活 | |
NCS+ | 174.32 | 41.34U/ml |
NCS+’ | 145.86 | 28.18U/ml |
NCS-wt | 117.64 | 18.26U/ml |
NCS-’ | 63.53 | 13.44U/ml |
NCS- | 9.41 | 5.83U/ml |
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (16)
- 一种调控普鲁兰酶表达量的方法,其特征在于,选取普鲁兰酶N端编码区前9n~10n个核苷酸,n=3,进行同义突变,构建突变体库,并计算P sfGFP值,根据P sfGFP值选择相应的同义突变序列;将目的蛋白的N端编码区进行相应突变,连接至表达载体,构建重组质粒;所述P sfGFP值按照下述方式计算:P sfGFP=274497.657-108717.401×GC3+4886.529×ΔG;所述GC3为编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸的同义密码子第三位碱基是GC的含量,n=3;所述ΔG为编码目的蛋白的基因的任意启动子转录起始位点至N端编码区的第90~99bp区域间的mRNA二级结构的最小自由能;普鲁兰酶表达量需要上调的时候,选择突变库中的P sfGFP值处于前10%的核苷酸序列;普鲁兰酶表达量需要下调的时候,选择突变库中的P sfGFP值处于后10%的核苷酸序列;将重组质粒导入枯草芽孢杆菌,利用枯草芽孢杆菌生产蛋白;所述普鲁兰酶的氨基酸序列如SEQ ID NO.19所示。
- 一种筛选编码不同表达量的蛋白的核苷酸序列的方法,其特征在于,测定GC3和ΔG的值,再应用下述方程式计算蛋白的相对表达量,即P sfGFP值,根据P sfGFP值筛选出对应的核苷酸序列;P sfGFP值与蛋白的实际表达量成正相关:P sfGFP=274497.657-108717.401×GC3+4886.529×ΔG;所述GC3为编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸的同义密码子第三位碱基是GC的含量,n=3;所述ΔG为目的基因的任意启动子转录起始位点至N端编码区的第90~99bp区域间的mRNA二级结构的最小自由能。
- 根据权利要求2所述的方法,其特征在于,所述ΔG为目的基因的任意启动子转录起始位点至N端编码区的第96bp区域间的mRNA二级结构的最小自由能。
- 根据权利要求3所述的方法,其特征在于,所述蛋白为能够在枯草芽孢杆菌中表达的任意蛋白。
- 根据权利要求4所述的方法,其特征在于,所述蛋白包括但不限于普鲁兰酶。
- 根据权利要求5所述的方法,其特征在于,所述普鲁兰酶的氨基酸序列如SEQ ID NO.19所示。
- 根据权利要求5所述的方法,其特征在于,根据P sfGFP值筛选相应的核苷酸序列。
- 一种调控基因工程菌蛋白表达量的方法,其特征在于,取目的蛋白N端编码区前9n~10n个核苷酸,n=3,建立同义突变库;计算同义突变库中的基因的参数GC3和ΔG,根据方程计算每个核苷酸序列的相对表达量,选择具有所需表达量的核苷酸序列,将目的蛋白N端编码区进行相应突变,并将其转化到宿主细胞中;所述方程为:P sfGFP=274497.657-108717.401×GC3+4886.529×ΔG;所述GC3为编码目的蛋白的基因靠近ATG的N端编码区前9n~10n个核苷酸的同义密码子第三位碱基是GC的含量,n=3;所述ΔG为目的基因的任意启动子转录起始位点至N端编码区的第90~99bp区域间的mRNA二级结构的最小自由能;蛋白表达量需要上调的时候,选择突变库中的P sfGFP值处于前10%的核苷酸序列;蛋白表达量需要下调的时候,选择突变库中的P sfGFP值处于后10%的核苷酸序列。所述ΔG为目的基因的任意启动子转录起始位点至N端编码区的第96bp区域间的mRNA二级结构的最小自由能。
- 根据权利要求8所述的方法,其特征在于,所述基因工程菌以枯草芽孢杆菌为宿主。
- 根据权利要求9所述的方法,其特征在于,所述蛋白为能够在枯草芽孢杆菌中表达的任意蛋白。
- 根据权利要求10所述的方法,其特征在于,所述蛋白包括但不限于普鲁兰酶。
- 根据权利要求11所述的方法,其特征在于,所述普鲁兰酶的氨基酸序列如SEQ ID NO.19所示。
- 一种调控普鲁兰酶表达量的方法,其特征在于,选取普鲁兰酶N端编码区前9n~10n个核苷酸,n=3,进行同义突变,构建突变体库,并计算P sfGFP值,根据P sfGFP值选择相应的同义突变序列;将目的蛋白的N端编码区进行相应突变,连接至表达载体,构建重组质粒;普鲁兰酶表达量需要上调的时候,选择突变库中的P sfGFP值处于前10%的核苷酸序列;普鲁兰酶表达量需要下调的时候,选择突变库中的P sfGFP值处于后10%的核苷酸序列。
- 根据权利要求13所述的方法,其特征在于,将重组质粒导入枯草芽孢杆菌,利用枯草芽孢杆菌生产蛋白。
- 根据权利要求14所述的方法,其特征在于,所述普鲁兰酶的氨基酸序列如SEQ ID NO.19所示。
- 权利要求1~12任一所述方法在调节目的蛋白表达量中的应用。
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