毕赤酵母中tRNA_(CCG)~(Pro)基因的表达及其作用效果
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摘要
转运核糖核酸(tRNA)是蛋白质合成过程中重要参与成分之一,为了探索稀有密码子对应的tRNA(稀少tRNA)丰度改变对外源基因表达量的影响,文中构建了毕赤酵母稀少tRNA基因与外源基因共表达体系。首先在GFP基因中添加由4个连续脯氨酸稀有密码子CCG组成的阻遏区,结果显示该GFP基因的表达量明显降低。然后将带有阻遏区的GFP基因和tRNA_(CCG)~(Pro)基因顺次连接于pPIC9K载体上,在毕赤酵母GS115中共表达,结果使GFP表达量提高了4.9%;另将带有阻遏区的GFP基因和tRNA_(CCG)~(Pro)基因分别连接于pPIC9K和pFLDα载体,在毕赤酵母GS115中共表达,GFP表达量最高提高了12.5%;应用同样方式将tRNA_(CCG)~(Pro)基因与NFATc3T-GFP融合基因共表达,其表达量提高了21.3%。可见,tRNA_(CCG)~(Pro)在毕赤酵母GS115中确为稀少tRNA,通过共表达tRNA_(CCG)~(Pro)基因可显著提高带有连续该密码子的外源基因表达量,并且,文中构建的共表达体系将同样适用于其他稀少t RNA基因的筛选和验证。
Translocation ribonucleic acid(tRNA) is one of the important components in protein synthesis.In order to explore the effect of the changes of tRNAs corresponding to rare codons(rarity tRNAs) on the expression of exogenous genes,the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed.The expression of GFP in P.pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene.The expression amount of the repressed GFP could be increased about 4.9% when tRNA_(CCG)~(Pro) gene was cointegrated to the 3′ of the repressed GFP gene through pPIC9 K to the genome of P.pastoris GS115.Meanwhile,theexpression amount of the repressed GFP increased about 12.5% by integrating the repressed GFP gene and tRNA_(CCG)~(Pro) gene to the genome of P.pastoris GS115 through pPIC9 K and pFLDα,respectively.Using the same method,NFATc3T-GFP fusion gene and tRNA_(CCG)~(Pro) gene were co-expressed in P.pastoris GS115 resulting in 21.3% increased of the expression amount of NFATc3T-GFP fusion protein.In conclusion,tRNA_(CCG)~(Pro) gene has been confirmed to be a kind of rare tRNAs in P.pastoris GS115.Through co-expression of tRNA_(CCG)~(Pro) gene and heterologous genes which containing the continuous rare codon CCG,the expression of the repressed heterologous genes could be increased significantly.Furthermore,this co-expression system would contribute to screening and determining the other rare tRNAs.
Translocation ribonucleic acid(tRNA) is one of the important components in protein synthesis.In order to explore the effect of the changes of tRNAs corresponding to rare codons(rarity tRNAs) on the expression of exogenous genes,the co-expression system of rare tRNA gene and exogenous gene in Pichia pastoris was constructed.The expression of GFP in P.pastoris can be greatly reduced when a repressor region composed of four continuous proline rare codon CCG was added into the GFP gene.The expression amount of the repressed GFP could be increased about 4.9% when tRNA_(CCG)~(Pro) gene was cointegrated to the 3′ of the repressed GFP gene through pPIC9 K to the genome of P.pastoris GS115.Meanwhile,theexpression amount of the repressed GFP increased about 12.5% by integrating the repressed GFP gene and tRNA_(CCG)~(Pro) gene to the genome of P.pastoris GS115 through pPIC9 K and pFLDα,respectively.Using the same method,NFATc3T-GFP fusion gene and tRNA_(CCG)~(Pro) gene were co-expressed in P.pastoris GS115 resulting in 21.3% increased of the expression amount of NFATc3T-GFP fusion protein.In conclusion,tRNA_(CCG)~(Pro) gene has been confirmed to be a kind of rare tRNAs in P.pastoris GS115.Through co-expression of tRNA_(CCG)~(Pro) gene and heterologous genes which containing the continuous rare codon CCG,the expression of the repressed heterologous genes could be increased significantly.Furthermore,this co-expression system would contribute to screening and determining the other rare tRNAs.
引文
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[23]Clarke IV TF,Clark PL.Rare codons cluster.PLoSONE,2008,3(10):e3412.
[24]Novoa EM,de Pouplana LR.Speeding with control:codon usage,tRNAs,and ribosomes.Trends Genet,2012,28(11):574-581.
[25]Shin CS,Min SH,Shin HC,et al.High-level production of recombinant human IFN-α2a with co-expression of t RNAArg(AGG/AGA)in high-cell-density cultures of Escherichia coli.Biotechnol Biopr Eng,2001,6(4):301-305.
[2]Fedyunin I,Lehnhardt L,B?hmer N,et al.tRNAconcentration fine tunes protein solubility.FEBSLett,2012,586(19):3336-3340.
[3]Feng DJ,Liu X,Li XG,et al.The relationship between tRNA abundance and gene expression.China Biotechnol,2002,22(6):4-8(in Chinese).冯德江,刘翔,李旭刚,等.tRNA丰度与基因表达的关系.中国生物工程杂志,2002,22(6):4-8.
[4]Quax TEF,Claassens NJ,S?ll D,et al.Codon bias as a means to fine-tune gene expression.Mol Cell,2015,59(2):149-161.
[5]Bierne N,Eyre-Walker A.Variation in synonymous codon use and DNA polymorphism within the Drosophila genome.J Evol Biol,2006,19(1):1-11.
[6]Burgess-Brown NA,Sharma S,Sobott F,et al.Codon optimization can improve expression of human genes in Escherichia coli:A multi-gene study.Prot Expr Purif,2008,59(1):94-102.
[7]Niu DD,Shen W,Wang ZX.Modification of intracellular aminoacyl-tRNA pool to improve expression level of exogenous genes in Escherichia coli.J Microbiol,2005,25(2):9-12(in Chinese).牛丹丹,沈微,王正祥.改善大肠杆菌胞内氨基酰tRNA池提高外源基因表达水平.微生物学杂志,2005,25(2):9-12.
[8]Cheng DH,Wang R,Prather KJ,et al.Tackling codon usage bias for heterologous expression in Rhodobacter sphaeroides by supplementation of rare tRNAs.Enzym Microb Technol,2015,72:25-34.
[9]Calderone TL,Stevens RD,Oas TG.High-level misincorporation of lysine for arginine at AGAcodons in a fusion protein expressed in Escherichia coli.J Mol Biol,1996,262(4):407-412.
[10]Aguirre-López B,Cabrera N,de Gómez-Puyou MT,et al.The importance of arginine codons AGA and AGG for the expression in E.coli of triosephosphate isomerase from seven different species.Biotechnol Rep,2017,13:42-48.
[11]Rosano GL,Ceccarelli EA.Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain.Microb Cell Fact,2009,8:41.
[12]Dieci G,Bottarelli L,Ballabeni A,et al.tRNA-assisted overproduction of eukaryotic ribosomal proteins.Prot Expr Purificat,2000,18(3):346-354.
[13]Kleber-Janke T,Becker WM.Use of modified BL21(DE3)Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage.Prot Expr Purificat,2000,19(3):419-424.
[14]Novy R,Drott D,Yaeger K,et al.Overcoming the codon bias of E.coli for enhanced protein expression.Sibirskmatzh,2001,3:613-623.
[15]Raval R,Simsa R,Raval K.Expression studies of Bacillus licheniformis chitin deacetylase in E.coli Rosetta cells.Int J Biol Macromol,2017,104:1692-1696.
[16]Yedahalli SS,Rehmann L,Bassi A.Expression of exo-inulinase gene from Aspergillus niger 12 in E.coli strain Rosetta-gami B(DE3)and its characterization.Biotechnol Prog,2016,32(3):629-637.
[17]Tegel H,Tourle S,Ottosson J,et al.Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3).Protein Expr Purif,2010,69(2):159-167.
[18]Finger C,Gamer M,Klunkelfu?S,et al.Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium.Appl Microbiol Biotechnol,2015,99(21):8999-9010.
[19]Lowe TM,Chan PP.tRNAscan-SE on-line:integrating search and context for analysis of transfer RNA genes.Nucleic Acids Res,2016,44(W1):W54-W57.
[20]Rogers HH,Griffiths-Jones S.tRNA anticodon shifts in eukaryotic genomes.RNA,2014,20(3):269-281.
[21]Lagunas L,Clipstone NA.Deregulated NFATc1activity transforms murine fibroblasts via an autocrine growth factor-mediated Stat3-dependent pathway.J Cellul Biochem,2009,108(1):237-248.
[22]Zhao H,Zhao SH.Progress of nuclear factor of activated T cells in cancer progression.Chin J Clin Electr Ed,2014,8(14):2696-2700(in Chinese).赵宏,赵守华.活化T细胞核因子与肿瘤的研究进展.中华临床医师杂志,2014,8(14):2696-2700.
[23]Clarke IV TF,Clark PL.Rare codons cluster.PLoSONE,2008,3(10):e3412.
[24]Novoa EM,de Pouplana LR.Speeding with control:codon usage,tRNAs,and ribosomes.Trends Genet,2012,28(11):574-581.
[25]Shin CS,Min SH,Shin HC,et al.High-level production of recombinant human IFN-α2a with co-expression of t RNAArg(AGG/AGA)in high-cell-density cultures of Escherichia coli.Biotechnol Biopr Eng,2001,6(4):301-305.