Eisinger, Dominic P

Trumpower, Bernard L

English

Dartmouth Digital Commons (Dartmouth College)

Dartmouth College Dartmouth Digital Commons Dartmouth Scholarship Faculty Work 1-1-1997 Long-inverse PCR to generate regional peptide libraries by codon mutagenesis Dominic P. Eisinger Dartmouth College Bernard L. Trumpower Dartmouth College Follow this and additional works at: https://digitalcommons.dartmouth.edu/facoa Dartmouth Digital Commons Citation Eisinger, Dominic P. and Trumpower, Bernard L., "Long-inverse PCR to generate regional peptide libraries by codon mutagenesis" (1997). Dartmouth Scholarship. 4249. https://digitalcommons.dartmouth.edu/facoa/4249 This Article is brought to you for free and open access by the Faculty Work at Dartmouth Digital Commons. It has been accepted for inclusion in Dartmouth Scholarship by an authorized administrator of Dartmouth Digital Commons. For more information, please contact dartmouthdigitalcommons@groups.dartmouth.edu. BenchmarksREFERENCES1.Beavis, R.C. and B.T. Chait. 1990. Rapid,sensitive analysis of protein mixtures by massspectrometry. Proc. Natl. Acad. Sci. USA87:6873-6877.2.Billeci, T.M. and J.T. Stults. 1993. Trypticmapping of recombinant proteins by matrix-assisted laser desorption/ionization mass spec-trometry. Anal. Chem. 65:1709-1716.3.Bornsen, K.O., M.D. Mohr and H.M. Wid-mar. 1995. Ion exchange and purification ofcarbohydrates on a Nafion® membrane as anew sample pretreatment for matrix-assistedlaser desorption/ionization mass spectrometry.Rapid Commun. Mass Spectrom. 9:1031-1034.4.Hutchens, T.W. and T.-T. Yip. 1993. Newdesorption strategies for the mass spectromet-ric analysis of macromolecules. Rapid Com-mun. Mass Spectrom. 7:576-580.5.Mock, K.K., C.W. Sutton and J.S. Cottrell.1992. Sample immobilization protocols formatrix-assisted laser-desorption mass spec-trometry. Rapid Commun. Mass Spectrom.6:233-238.6.Moritz, R., J. Eddes, H. Ji, G.E. Reid andR.J. Simpson. 1995. Rapid separation of pro-teins and peptides using conventional silica-based supports: identification of 2-D gel pro-teins following in-gel proteolysis, p. 311-319.In J.W. Crabb (Ed.), Techniques in ProteinChemistry VI. Academic Press, San Diego.7.Ortiz, M.L., M. Calero, C.F. Patron, L.Castellanos and E. Mendez. 1992. Imida-zole-SDS-Zn reverse staining of proteins ingels containing or not SDS and microse-quence of individual unmodified electroblot-ted proteins. FEBS Lett. 296:300-304.8.Patterson, S.D. 1995. Matrix-assisted laser-desorption/ionization mass spectrometric ap-proaches for the identification of gel-separat-ed proteins in the 5–50 pmol range.Electrophoresis 16:1104-1114.9.Patterson, S.D. and V. Katta. 1994. Promptfragmentation of disulfide-linked peptidesduring matrix-assisted laser desorption ioniza-tion mass spectrometry. Anal. Chem.66:3727-3732.10.Shevchenko, A., M. Wilm, O. Vorm and M.Mann. 1996. Mass-spectrometric sequencingof proteins from silver-stained polyacrylamidegels. Anal. Chem. 68:850-858.11.Vorm, O., P. Roepstorff and M. Mann.1994. Improved resolution and very high-sen-sitivity in MALDI TOF of matrix surfacesmade by fast evaporation. Anal. Chem.66:3281-3287.12.Xiang, F. and R.C. Beavis. 1994. A methodto increase contaminant tolerance in proteinmatrix-assisted laser desorption/ionization bythe fabrication of thin protein-doped polycrys-talline films. Rapid Commun. Mass Spectrom.8:199-204.13.Zaluzec, E.J., D.A. Gage, J. Allison and J.T.Watson. 1994. Direct matrix-assisted laser-desorption ionization mass-spectrometricanalysis of proteins immobilized on nylon-based membranes. J. Am. Soc. Mass Spec-trom. 5:230-237.14.Zhang, H., P.E. Andren and R.M. Caprioli.1995. Micro-preparation procedure for high-sensitivity matrix-assisted laser desorptionionization mass spectrometry. J. Mass Spec-trom. 30:1768-1771.We would like to thank Dr. R. Rush forhis initial suggestion for the use of thesecolumns and reading of the manuscript andDrs. Hsieng Lu and Tony Polverino for crit-ical reading of the manuscript. We also wishto thank Scott Lauren for amino acid analy-ses. Address correspondence to Scott D.Patterson, Amgen, Amgen Center, Mail Stop14-2-E, 1840 DeHavilland Drive, ThousandOaks, CA 91320-1789, USA. Internet: spatters@amgen.comReceived 1 May 1996; accepted 26 July1996.Paul L. Courchesne and ScottD. PattersonAmgenThousand Oaks, CA, USALong-Inverse PCR ToGenerate Regional Peptide Libraries ByCodon MutagenesisBioTechniques 22:250-254 (February 1997)We have developed a method to con-struct protein libraries by codon muta-genesis, which can be applied to anyplasmid-encoded gene without the needfor appropriate restriction sites and sub-cloning. The procedure entails synthe-sis of two oligonucleotides, in one ofwhich selected codons are partially ran-domized, alkaline denaturation of tem-plate plasmid followed by long-inversepolymerase chain reaction (PCR) andtreatment of DNA termini for blunt-endself-ligation to generate a mutagenizedplasmid library.Changes of amino acids by mutage-nesis of cloned genes is increasinglyused to examine structure-function re-lationships in proteins. Though useful,site-directed oligonucleotide mutagen-esis is labor-intensive when manychanges are required. Random mutage-nesis of plasmids with chemical agentsor E. coli mutator strains generates mu-tations outside of the gene of interestand becomes increasingly impracticalas the size ratio of the plasmid to targetgene increases. Procedures in which thetarget gene is amplified by PCR underconditions that increase the mis-incor-poration rate by Taq DNA polymerasenarrow the mutagenized region to thearea of interest, but require high-effi-ciency cloning of the product and suit-able restriction sites in both the productand target vector to generate a libraryof sufficient size (10). In addition, thesemethods are biased for transition muta-tions over transversions, which limitsthe amino acid substitutions at any giv-en position. Furthermore, the geneticcode itself minimizes the harmful ef-fects of mutations (9). For example,pyrimidines in the second codon posi-tion specify predominately hydropho-bic amino acids. An additional limita-tion in obtaining a broad spectrum ofamino acid substitutions at a givencodon is the extremely rare occurrenceof an adjacent double-point mutation.Codon-based mutagenesis or region-250 BioTechniques Vol. 22, No. 2 (1997)Benchmarksal codon randomization generates high-ly complex yet condensed protein li-braries (2,4). This procedure relies oncomplex oligonucleotide synthesis inwhich a portion of the appropriatecodons are randomized using all 64codons or a subset of codons on oneDNA synthesis column while the wild-type codon is synthesized on anothercolumn. The matrices from the twocolumns are mixed and split again forthe next randomized codon, and theprocess is repeated for each random-ized codon. The mutation frequency isset by the ratio of resin in the columncontaining the mutated codon vs. thatcontaining the wild-type codon and isbiased to the wild-type sequence. Typi-cally, conditions are chosen such thatfor a 20-amino acid sequence, 3000clones will contain all possible singleamino acid substitutions in addition tomultiple substitutions at more than oneposition (2). The technique is not yet inwidespread use, partly because of diffi-culties in generating a representative li-brary and the necessity of introducingspecific restriction enzyme sites. We were recently unable to isolatetemperature-sensitive mutations in theessential Saccharomyces cerevisiaegene QSR1 (11) by random mutagene-sis procedures, which were successfulin our hands with a gene of similar size(5). The possibility exists that a temper-ature-sensitive QSR1 allele was not ob-tained since not all proteins can be ren-dered thermolabile by the limitedamino acid substitutions possible bysingle mutations (7). We thus devel-oped a simple method of regionalcodon randomization mutagenesis. Wetargeted the mutagenesis to a highlycharged 20-amino acid sequence on thepremise that clusters of charged resi-dues are likely to be on the surface ofthe protein and may be important forprotein-protein interactions (12). Withthis procedure, we obtained 25 uniquetemperature-sensitive mutants from asingle library.The procedure is outlined in Figure1 and simplifies randomization or site-directed mutagenesis of any region onany sized plasmid without the need forrestriction enzymes. Long PCR (1) oftwo primers back to back (inversePCR) (8) forms an 8-kb fragment of theentire plasmid. Long PCR allows forhigh-yield amplification of fragmentsin the 6–25-kb range and has the addedbenefit that fidelity is increased severalfold because of the addition of a proof-reading thermostable DNA polyme-rase. The fragment is treated with T4DNA polymerase to remove 3′ over-hanging A’s added by Taq polymerase,then phosphorylated and self-ligated toregenerate a plasmid library incorporat-ing the oligonucleotide mixture.Three micrograms of the 8-kb plas-mid pDEQ2 were denatured in 200 µLof 0.2 M NaOH and 2 mM EDTA for10 min at 37°C, then precipitated at -20°C by adding 20 µL of 3 M Naacetate (pH 5.2) and 700 µL of ethanol.The pellet was washed and resuspend-ed in 300 µL of water at a final concen-tration of 10 ng/µL. Alkaline denatura-tion before PCR increases productyield and eliminates unwanted back-ground transformants from the tem-plate plasmid (3).The Expand Long-Template PCRsystem (Boehringer Mannheim, Indi-anapolis, IN, USA) was used to gener-ate a plasmid library incorporating the20-amino acid randomized codon re-gion. A partially randomized 81-nu-cleotide (nt) oligonucleotide was syn-thesized (2). A titration of MgCl2concentration was carried out by usingall 3 supplied 10× buffers, and bufferNo. 2 (2.25 mM MgCl2) was chosen onthe basis of a high product yield. All252 BioTechniques Vol. 22, No. 2 (1997)Figure 1. Mutagenesis by long-inverse PCR. Primers were designed in a back-to-back configuration(inverse PCR) such that self-ligation of the PCR product regenerates the template plasmid. The C1primer is 21-nt long, the R1 is 81 nt in length with the 5′ 60 nt consisting of a mixture of wild-type andrandomized codons (hatched area), while the 3′ 21 nt are wild-type for efficient annealing. The four stepsare: 1. Long-inverse PCR with the two primers, 2. removal of 3′ overhanging A’s with T4 DNA poly-merase, 3. phosphorylation of 5′ termini and 4. self-ligation to regenerate a plasmid library incorporatingthe randomized oligonucleotide pool.Benchmarkscomponents were mixed on ice, and a200-µL reaction was split into 4 thin-walled 0.6-mL PCR tubes (RobbinsScientific, Sunnyvale, CA, USA). Finalconcentrations were 350 µM dNTPs,1× PCR buffer, 1.5 µL of enzyme mix-ture and 200 nM for each primer. Afteroverlaying with mineral oil and placingin a thermal cycler preheated to 94°C,0.2 ng of denatured template in 2 µLwas added to each tube. Cycling condi-tions were 94°C for 10 s, 50°C for 2min and 68°C for 6 min for 10 cyclesfollowed by 15 cycles with a 20-s ex-tension successively added to each68°C extension step. At the end of 25cycles, an additional 68°C extensionfor 10 min was included before coolingto 4°C. Depending upon the primerpairs, yields ranged from 1–10 µg ofproduct.The PCRs were pooled with 200 µLof 2× proteinase K buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mMEDTA, 1% sodium dodecyl sulfate) and20 µg of proteinase K (BoehringerMannheim) and incubated for 30 min at37°C. After successive phenol, phe-nol/chloroform and chloroform extrac-tions, the DNA product was precipitat-ed with 1 mL of ethanol at -70°C for 10min. After washing with 70% ethanoland air drying, the pellet was resus-pended in water and blunted with 4 U ofT4 DNA polymerase (BoehringerMannheim) in the presence of 80 µMdNTPs for 2 h at 16°C. To changebuffers, the DNA was purified byGENECLEAN® (BIO 101, Vista, CA,USA) and eluted in 16 µL of water. Tothis, 2 µL of 10× polynucleotide kinasebuffer, 1 µL of 2 mM ATP and 1 µL (10U) of polynucleotide kinase (Boeh-ringer Mannheim) were added. After in-cubation at 37°C for 1 h, the 8-kb prod-uct band was gel-purified on a 0.7%agarose gel in 40 mM Tris acetate, 2mM EDTA, pH 8.5, and eluted at a con-centration of approximately 10 ng/µL,assuming a 50% loss with all manipula-tions. The kinasing step can be omittedif the primers are phosphorylated.A self-ligation with 60 ng of DNAin a volume of 10 µL with 40 U of T4ligase (New England Biolabs, Beverly,MA, USA) was carried out for 16 h at16°C. Transformation competentDH5α cells were prepared by the high-efficiency procedure of Hanahan (6),and 8000 individual colonies were ob-tained per 5 µL of ligation transformed,which is greater than 105 clones per µgof DNA. A control lacking ligase gavea negligible background of 4 colonies.A library was made by pooling 18 000colonies and purifying the plasmidDNA. Sequence analysis of 30 randomclones revealed an average of 1.08 mu-tated codons per clone, whereas the 25temperature-sensitive clones isolatedhad an average of 1.8 mutated codonsper clone and were primarily single anddouble amino acid substitutions with afew triple substitutions. This higherrate of multiple mutated codons and theobservation that only unique substitu-tions were obtained at a given codon re-inforce the hypothesis that some genesare not rendered thermolabile by sin-gle-point mutations.We have described a method oflong-inverse PCR to make compactpeptide libraries by codon mutagenesison any plasmid. The procedure is alsoapplicable to other gene modifications,and we have successfully used it forsite-directed mutagenesis, protein trun-cations and epitope tagging.REFERENCES1.Barnes, W. 1994. PCR amplification of up to35-kb DNA with high fidelity and high yieldfrom λ bacteriophage templates. Proc. Natl.Acad. Sci. USA 91:2216-2220.2.Cormack, B.P. and K. Struhl. 1993. Region-al codon randomization: defining a TATA-binding protein surface required for RNApolymerase III transcription. Science 262:244-248.3.Du, Z., D.A. Regier and R.C. Desrosiers.1995. Improved recombinant PCR mutagene-sis procedure that uses alkaline-denaturedplasmid template. BioTechniques 18:376-378.4.Glasser, S.M., D.E. Yelton and W.D. Huse.1992. Antibody engineering by codon-basedmutagenesis in a filamentous phage vectorsystem. J. Immunol. 149:3903-3913.5.Graham, L.A., U. Brandt, J.S. Sargent andB.L. Trumpower. 1992. Mutational analysisof assembly and function of the iron-sulfurprotein of the cytochrome bc1 complex inSaccharomyces cerevisiae. J. Bioenerg. Bio-membr. 25:245-257.6.Hanahan, D. 1985. Techniques for transfor-mation of E. coli, p. 115. In D.M. Glover(Ed.), DNA Cloning-A Practical Approach.Vol. 1. IRL Press, Oxford.7.Harris, S.D., J. Cheng, T. Pugh and J.R.Pringle. 1992. Molecular analysis of Saccha-romyces cerevisiae chromosome I: on thenumber of genes and the identification of es-sential genes using temperature-sensitive-lethal mutations. J. Mol. Biol. 225:53-65.8.Hemsley, A., N. Arnheim, M.D. Toney, G.Cortopassi and D.J. Galas. 1989. A simplemethod for site-directed mutagenesis usingthe polymerase chain reaction. Nucleic AcidsRes. 16:6545-6551.9.Lagerkvist, U. 1981. Unorthodox codon read-ing and the evolution of the genetic code. Cell23:305-306.10.Leung, D.W., E. Chen and D.V. Goeddel.1989. A method for random mutagenesis of adefined segment using a modified polymerasechain reaction. Techniques 1:11-15.11.Tron, T., M. Yang, F.A. Dick, M.E. Schmittand B.L. Trumpower. 1995. QSR1, an essen-tial yeast gene with a genetic relationship to asubunit of the mitochondrial cytochrome bc1complex, is homologous to a gene implicatedin eukaryotic cell differentiation. J. Biol.Chem. 270:9961-9970.12.Wertman, K.F., D.G. Drubin and D. Bot-stein. 1992. Systematic mutational analysis ofthe yeast ACT1 gene. Genetics 132:337-350.This work was supported by Grant No.BE-204 from the American Cancer Society.Address correspondence to Dominic P.Eisinger, Dartmouth Medical School, Vail7200, Hanover, NH 03755, USA.Received 3 June 1996; accepted 22August 1996.Dominic P. Eisinger andBernard L. TrumpowerDartmouth Medical SchoolHanover, NH, USAFamily of Plasmid Vectorsfor the Expression of β-galactosidase Fusion Pro-teins in Eukaryotic CellsBioTechniques 22:254-258 (February 1997)LacZ is a popular reporter gene for avariety of biochemical and genetic ana-lyses. The enzymatic activity of its pro-tein product, β-galactosidase, can beeasily measured by well-developedspectrophotometric (4) and luminomet-ric (1) assays in vitro and by histo-chemical staining in vivo (10). More-over, it is possible to follow the spatialdistribution of the gene product in situ,even in living animals, using lipophilicsubstrates (7,11). Antibodies against β-galactosidase made it possible to254 BioTechniques Vol. 22, No. 2 (1997)

Long-inverse PCR to generate regional peptide libraries by codon mutagenesis

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Long-inverse PCR to generate regional peptide libraries by codon mutagenesis

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