芳香硫酸酯酶定点饱和突变方案的比较
|
摘要
目的比较随机引物与精确引物PCR扩增实施绿脓杆菌芳香硫酸酯酶(Pseudomonas aeruginosa arylsulfatase, PAAS)定点饱和突变的整体效率及成本,以建立高效实用的定点饱和突变方案。方法以大肠杆菌碱性磷酸酶(Escherichia coli alkaline phosphatase, ECAP)与PAAS融合表达质粒为模板,分别用针对PAAS目的位点的随机引物、精确引物等量混合物及单个精确引物,进行全质粒PCR扩增构建定点饱和突变体库;碱裂解细胞后高通量测定PAAS突变体与ECAP活性比值进行筛选,比较3种定点饱和突变建库方案需筛选的单克隆数量、所获突变体种类、整体耗时、整体成本及影响因素。结果用随机引物PCR对M72位定点饱和突变有明显密码子偏好性,筛选超600个单克隆仅获得10种预期突变体,但对G138位定点饱和突变未见明显的密码子偏好性且筛选不到150个单克隆获得18种预期突变体;精确引物等量混合物对M72位实施定点饱和突变,筛选不超过190个克隆成功获得19种预期突变体;单个精确引物对M72位逐个PCR实施定点饱和突变,仅各筛选2个单克隆就获得19种预期突变体。单个精确引物逐个PCR实施定点突变所获饱和突变体库完整,需筛选的单克隆数最少,总耗时和总成本最低;相比之下,用随机引物和精确引物等量混合物实施定点饱和突变时,所需筛选的单克隆数、整体耗时及成本均显著增加。结论解析序列-活性关系时,用单个精确引物逐个PCR实施定点饱和突变有显著整体成本和效率优势;仅筛选阳性突变体时,用精确引物等量混合物进行PCR建库在成本和效率上更有优势。
Objective To compare the overall efficiency and cost of the construction and screening of libraries of Pseudomonas aeruginosa arylsulfatase(PAAS) through site-directed saturation mutagenesis with random primers(NNN) and precise primers for PCR. Methods Site-directed mutagenesis libraries were constructed using the random primers(NNN) of the selected site, an equal-mole mixture of precise primers and a purified precise primer each time for the amplification of the fusion vector of Escherichia coli alkaline phosphatase(ECAP) and PAAS through PCR. The libraries were screened in a high-throughput mode through the assay of activity ratios of PAAS/mutants to ECAP after alkaline lysis of host cells. Three approaches for site-directed saturation mutagenesis were compared for their number of monoclones screened, the number of their expected mutants discovered, overall time consumed, overall cost and other pertinent factors. Results The site-directed saturation mutagenesis of M72 with random primers for PCR resulted in only 10 among 20 expected mutants after the screening of over 600 monoclones,besides obvious codon bias.In contrast,no obvious codon bias was observed and there were already 18 among 20 expected mutants after the screening of less than 150 monoclones for site-directed saturation mutagenesis of G138 with similar random primers.The site-directed saturation mutagenesis of M72 with an equal-mole mixture of precise primers yielded all of the 19 expected mutants after the screening of less than 190 monoclones;the site-directed saturation mutagenesis of M72 with the purified precise primers for PCR one-by-one gave all of the 19 expected mutants after the screening of just 2 monoclones for every expected mutant.The use of the purified precise primers for PCR one-by-one was thus more favorable for the construction of libraries of site-directed saturation mutagenesis for the minimum number of monoclones screened,the least overall time and cost.In comparison to the use of the purified precise primers for PCR one-by-one,the use of random primers or the equal-mole mixture of precise primers for PCR to generate the libraries tolerated much greater overall cost and longer time.Conclusion The generation and screening of the site-directed saturation mutagenesis libraries with precise primers for PCR one-by-one were more practical in elucidating the sequence-activity relationship while the use of equal-mole mixture of precise primers for PCR was preferable for the screening of positive mutants during site-directed saturation mutagenesis.
Objective To compare the overall efficiency and cost of the construction and screening of libraries of Pseudomonas aeruginosa arylsulfatase(PAAS) through site-directed saturation mutagenesis with random primers(NNN) and precise primers for PCR. Methods Site-directed mutagenesis libraries were constructed using the random primers(NNN) of the selected site, an equal-mole mixture of precise primers and a purified precise primer each time for the amplification of the fusion vector of Escherichia coli alkaline phosphatase(ECAP) and PAAS through PCR. The libraries were screened in a high-throughput mode through the assay of activity ratios of PAAS/mutants to ECAP after alkaline lysis of host cells. Three approaches for site-directed saturation mutagenesis were compared for their number of monoclones screened, the number of their expected mutants discovered, overall time consumed, overall cost and other pertinent factors. Results The site-directed saturation mutagenesis of M72 with random primers for PCR resulted in only 10 among 20 expected mutants after the screening of over 600 monoclones,besides obvious codon bias.In contrast,no obvious codon bias was observed and there were already 18 among 20 expected mutants after the screening of less than 150 monoclones for site-directed saturation mutagenesis of G138 with similar random primers.The site-directed saturation mutagenesis of M72 with an equal-mole mixture of precise primers yielded all of the 19 expected mutants after the screening of less than 190 monoclones;the site-directed saturation mutagenesis of M72 with the purified precise primers for PCR one-by-one gave all of the 19 expected mutants after the screening of just 2 monoclones for every expected mutant.The use of the purified precise primers for PCR one-by-one was thus more favorable for the construction of libraries of site-directed saturation mutagenesis for the minimum number of monoclones screened,the least overall time and cost.In comparison to the use of the purified precise primers for PCR one-by-one,the use of random primers or the equal-mole mixture of precise primers for PCR to generate the libraries tolerated much greater overall cost and longer time.Conclusion The generation and screening of the site-directed saturation mutagenesis libraries with precise primers for PCR one-by-one were more practical in elucidating the sequence-activity relationship while the use of equal-mole mixture of precise primers for PCR was preferable for the screening of positive mutants during site-directed saturation mutagenesis.
引文
[1] DAVIDS T,SCHMIDT M,BOTTCHER D,et al.Strategies for the discovery and engineering of enzymes for biocatalysis[J].Curr Opin Chem Biol,2013,17(2):215-220.
[2] REETZ MT,PRASAD S,CARBALLEIRA JD,et al.Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity:Rigorous comparison with traditional methods[J].J Am Chem Soc,2010,132(26):9144-9152.
[3] 陶苏丹,刘佳,陈喜文,等.点饱和突变技术及其在蛋白质工程中的应用[J].中国生物工程杂志,2007,27(8):82-86.
[4] REETZ MT,CARBALLEIRA JD.Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes[J].Nat Protoc,2007,2(4):891-903.
[5] CHOCKALINGAM K,CHEN Z,KATZENELLENBOGEN JA,et al.Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches[J].Proc Natl Acad Sci USA,2005,102(16):5691-5696.
[6] NAIR NU,ZHAO H.Evolution in reverse:engineering a D-xylose-specific xylose reductase[J].Chembiochem,2008,9(8):1213-1215.
[7] PRASAD S,BOCOLA M,REETZ MT.Revisiting the lipase from Pseudomonas aeruginosa:Directed evolution of substrate acceptance and enantioselectivity using iterative saturation mutagenesis[J].Chemphyschem,2011,12(8):1550-1557.
[8] REETZ MT,WANG LW,BOCOLA M.Directed evolution of enantioselective enzymes:iterative cycles of CASTing for probing protein-sequence space[J].Angew Chem Int Ed Engl,2006,45(8):1236-1241.
[9] HOGREFE HH,CLINE J,YOUNGBLOOD GL,et al.Creating randomized amino acid libraries with the QuikChange Multi Site-Directed Mutagenesis Kit[J].Bio Techniques,2002,33(5):1158-1160,1162,1164-1165.
[10] MAHANT NK.Chemiluminescence assays based on indoxyl substrates or thioindoxyl substrates:EP,EP 0775215 B1[P].2001.
[11] PARK JY,KRICKA LJ.Chemiluminescence enhanced detection:US,US 20110124965 A1[P].2011.
[12] WANG X,DUAN D,XU J,et al.Characterization of a novel alkaline arylsulfatase from Marinomonas sp.FW-1 and its application in the desulfation of red seaweed agar[J].J Ind Microbiol Biotechnol,2015,42(10):1353-1362.
[13] LIU H,YANG X,LIU L,et al.Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution:chemometrics and experimental models[J].Anal Chem,2013,85(4):2143-2154.
[14] LIU H,YUAN M,YANG X,et al.Comparison of candidate pairs of hydrolytic enzymes for spectrophotometric-dual-enzyme-simultaneous-assay[J].Anal Sci,2015,3 (5):421-427.
[15] BOLTES I,CZAPINSKA H,KAHNERT A,et al.1.3 A structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family[J].Structure,2001,9(6):483-491.
[16] 李雨薇,袁梅,杨晓兰,等.绿脓杆菌芳香硫酸酯酶序列与活性关系的初步表征[J].重庆医科大学学报,2018,43(1):107-112.
[17] HUGHES MD,NAGEL DA,SANTOS AF,et al.Removing the redundancy from randomised gene libraries[J].J Mol Biol,2003,331(5):973-979.
[18] YUAN M,YANG X,LI Y,et al.Facile alkaline lysis of Escherichia coli cells in high-throughput mode for screening enzyme mutants:Arylsulfatase as an example[J].Appl Biochem Biotechnol,2016,179(4):545-557.
[19] SAMBROOK J,FRITSCH EF,MANIATIS T,et al.Molecular Cloning:A Laboratory Manual[M].2nd ed.New York:Cold Spring Harbor Laboratory Press,1989.
[20] AIRD D,ROSS MG,CHEN WS,et al.Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries[J].Genome Biol,2011,12(2):R18.
[21] PALFREY D,PICARDO M,HINE AV,et al.A new randomization assay reveals unexpected elements of sequence bias in model ‘randomized' gene libraries:implications for biopanning[J].Gene,2000,251 (1):91-99.
[22] NEYLON C.Chemical and biochemical strategies for the randomization of protein encoding DNA sequences:Library construction methods for directed evolution[J].Nucleic Acids Res,2004,32 (4):1448-1459.
[23] YANG X,FENG Y,CHONG H,et al.High-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins[J].Anal Biochem,2017,534:91-98.
[2] REETZ MT,PRASAD S,CARBALLEIRA JD,et al.Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity:Rigorous comparison with traditional methods[J].J Am Chem Soc,2010,132(26):9144-9152.
[3] 陶苏丹,刘佳,陈喜文,等.点饱和突变技术及其在蛋白质工程中的应用[J].中国生物工程杂志,2007,27(8):82-86.
[4] REETZ MT,CARBALLEIRA JD.Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes[J].Nat Protoc,2007,2(4):891-903.
[5] CHOCKALINGAM K,CHEN Z,KATZENELLENBOGEN JA,et al.Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches[J].Proc Natl Acad Sci USA,2005,102(16):5691-5696.
[6] NAIR NU,ZHAO H.Evolution in reverse:engineering a D-xylose-specific xylose reductase[J].Chembiochem,2008,9(8):1213-1215.
[7] PRASAD S,BOCOLA M,REETZ MT.Revisiting the lipase from Pseudomonas aeruginosa:Directed evolution of substrate acceptance and enantioselectivity using iterative saturation mutagenesis[J].Chemphyschem,2011,12(8):1550-1557.
[8] REETZ MT,WANG LW,BOCOLA M.Directed evolution of enantioselective enzymes:iterative cycles of CASTing for probing protein-sequence space[J].Angew Chem Int Ed Engl,2006,45(8):1236-1241.
[9] HOGREFE HH,CLINE J,YOUNGBLOOD GL,et al.Creating randomized amino acid libraries with the QuikChange Multi Site-Directed Mutagenesis Kit[J].Bio Techniques,2002,33(5):1158-1160,1162,1164-1165.
[10] MAHANT NK.Chemiluminescence assays based on indoxyl substrates or thioindoxyl substrates:EP,EP 0775215 B1[P].2001.
[11] PARK JY,KRICKA LJ.Chemiluminescence enhanced detection:US,US 20110124965 A1[P].2011.
[12] WANG X,DUAN D,XU J,et al.Characterization of a novel alkaline arylsulfatase from Marinomonas sp.FW-1 and its application in the desulfation of red seaweed agar[J].J Ind Microbiol Biotechnol,2015,42(10):1353-1362.
[13] LIU H,YANG X,LIU L,et al.Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution:chemometrics and experimental models[J].Anal Chem,2013,85(4):2143-2154.
[14] LIU H,YUAN M,YANG X,et al.Comparison of candidate pairs of hydrolytic enzymes for spectrophotometric-dual-enzyme-simultaneous-assay[J].Anal Sci,2015,3 (5):421-427.
[15] BOLTES I,CZAPINSKA H,KAHNERT A,et al.1.3 A structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family[J].Structure,2001,9(6):483-491.
[16] 李雨薇,袁梅,杨晓兰,等.绿脓杆菌芳香硫酸酯酶序列与活性关系的初步表征[J].重庆医科大学学报,2018,43(1):107-112.
[17] HUGHES MD,NAGEL DA,SANTOS AF,et al.Removing the redundancy from randomised gene libraries[J].J Mol Biol,2003,331(5):973-979.
[18] YUAN M,YANG X,LI Y,et al.Facile alkaline lysis of Escherichia coli cells in high-throughput mode for screening enzyme mutants:Arylsulfatase as an example[J].Appl Biochem Biotechnol,2016,179(4):545-557.
[19] SAMBROOK J,FRITSCH EF,MANIATIS T,et al.Molecular Cloning:A Laboratory Manual[M].2nd ed.New York:Cold Spring Harbor Laboratory Press,1989.
[20] AIRD D,ROSS MG,CHEN WS,et al.Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries[J].Genome Biol,2011,12(2):R18.
[21] PALFREY D,PICARDO M,HINE AV,et al.A new randomization assay reveals unexpected elements of sequence bias in model ‘randomized' gene libraries:implications for biopanning[J].Gene,2000,251 (1):91-99.
[22] NEYLON C.Chemical and biochemical strategies for the randomization of protein encoding DNA sequences:Library construction methods for directed evolution[J].Nucleic Acids Res,2004,32 (4):1448-1459.
[23] YANG X,FENG Y,CHONG H,et al.High-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins[J].Anal Biochem,2017,534:91-98.