MutS, Thermus Aquaticus DNA Mismatch Repair Protein, DNA mismatch repair protein MutS.
The MutS DNA Mismatch Protein Identifies heteroduplex DNAs comprising mis-paired or unpaired bases. The MutS DNA Mismatch Protein binds in vitro to heteroduplex DNAs comprising mis-paired or unpaired bases over a varied temperature range from 4-70 °C and obtains a thermostable ATPase activity. The MutS DNA Mismatch Protein is active at temperature between 0 to 75°C. Since The MutS DNA Mismatch Protein competently binds to 1-4 bases deletion (or insertion) and mismatch base pairs of GT, CT and AG, it is suitable for sensing these mutations. Mutations can be distinguished in polyacrylamide gels or on a solid phase such as Ni agarose or beads or magnetic Ni-NTA particles.
DNA Mismatch Repair Protein MutS Thermus Aquaticus Recombinant produced in E.coli is a single, non-glycosylated polypeptide chain containing 829 amino acids and having a molecular mass of 92.8kDa.
The Thermus Aquaticus is fused to a 6 amino acid His-Tag at C-terminus and purified by proprietary chromatographic techniques.
The MutS protein is supplied in 20mM Tris-HCl, pH-8, 250mM NaCl, 0.1mM EDTA, 1mM DTT and 50% Glycerol.
100mM KCl, 50mM Tris-HCl, pH-8.5, 20mM MgCl2, 0.1mM EDTA, 1mM DTT and 2% Glycerol, 65 °C.
Greater than 95.0% as determined by SDS-PAGE.
Amino acid sequence
MKIEHMEGML KGEGPGPLPP LLQQYVELRD QYPDYLLLFQ VGDFYECFGE DAERLARALG LVLTHKTSKD FTTPMAGIPL RAFEAYAERL LKMGFRLAVA DQVEPAEEAE GLVRREVTQL LTPGTLLQES LLPREANYLA AIATGDGWGL AFLDVSTGEF KGTVLKSKSA LYDELFRHRP AEVLLAPELL ENGAFLDEFR KRFPVMLSEA PFEPEGEGPL ALRRARGALL AYAQRTQGGA LSLQPFRFYD PGAFMRLPEA TLRALEVFEP LRGQDTLFSV LDETRTAPGR RLLQSWLRHP LLDRGPLEAR LDRVEGFVRE GALREGVRRL LYRLADLERL ATRLELGRAS PKDLGALRRS LQILPELRAL LGEEVGLPDL SPLKEELEAA LVEDPPLKVS EGGLIREGYD PDLDALRAAH REGVAYFLEL EERERERTGI PTLKVGYNAV FGYYLEVTRP YYERVPKEYR PVQTLKDRQR YTLPEMKEKE REVYRLEALI RRREEEVFLE VRERAKRQAE ALREAARILA ELDVYAALAE VAVRYGYVRP RFGDRLQIRA GRHPVVERRT EFVPNDLEMA HELVLITGPN MAGKSTFLRQ TALIALLAQV GSFVPAEEAH LPLFDGIYTR IGASDDLAGG KSTFMVEMEE VALILKEATE NSLVLLDEVG RGTSSLDGVA IATAVAEALH ERRAYTLFAT HYFELTALGL PRLKNLHVAA REEAGGLVFY HQVLPGPASK SYGVEVAAMA GLPKEVVARA RALLQAMAAR REGALDAVLE RLLALDPDRL TPLEALRLLQ ELKALALGAP LDTMKGKLAA ALEHHHHHH.
1. After first round PCR, purify PCR fragments using Qiagen QIAquick PCR purification kit with elution in dH2O.
2. Dilute PCR product to 250 ng/µl in 10 mM Tris–HCl, pH 7.8, 50 mM NaCl and heat to 95 °C for 5 min followed by cooling at 0.1°C /s to 25 °C.
3. Add binding buffer (20 mM Tris–HCl, pH 7.8, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT and 5% glycerol) to annealed PCR product, adjust DNA concentration to 11.5 ng/µl, add MutS dimers to 950 nM.
4. Incubate the mixture at room temperature for 10 min, then add an equal volume of Ni-NTA beads pre-equilibrated with 1×binding buffer, and incubate for 30 min at room temperature.
5. Gently spin down beads and save supernatant for subsequent processing (second round PCR, cloning etc).
1. Remove mismatch DNA (error correction) from gene synthesis reaction
2. Mutation detection and removal
3. Rapid isothermal SNP detection