MutS

Thermus Aquaticus DNA Mismatch Repair Protein MutS Recombinant

Cat. No.

BT2323

Source

Escherichia Coli.

Synonyms

MutS, Thermus Aquaticus DNA Mismatch Repair Protein, DNA mismatch repair protein MutS.

Appearance

Sterile filtered colorless solution.

Purity

Greater than 95.0% as determined by SDS-PAGE.

Usage

Prospec's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.

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Description

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.

Product Specs

Introduction

MutS DNA Mismatch Protein identifies heteroduplex DNA containing mispaired or unpaired bases. It binds to these heteroduplex DNAs in vitro over a temperature range of 4-70°C and exhibits thermostable ATPase activity. Active between 0-75°C, MutS effectively binds to 1-4 base deletions, insertions, and mismatches like GT, CT, and AG, making it suitable for detecting such mutations. These mutations can be analyzed through polyacrylamide gel electrophoresis or solid-phase methods utilizing substrates like Ni agarose, beads, or magnetic Ni-NTA particles.

Description

DNA Mismatch Repair Protein MutS from Thermus Aquaticus, recombinantly produced in E. coli, is a non-glycosylated polypeptide chain. It consists of 829 amino acids, resulting in a molecular weight of 92.8 kDa. A 6-amino acid His-Tag is fused to the C-terminus of the Thermus Aquaticus protein, which is then purified using proprietary chromatographic methods.

Physical Appearance

Clear, colorless solution, sterile-filtered.

Formulation

The MutS protein solution is formulated in 20mM Tris-HCl at pH 8, 250mM NaCl, 0.1mM EDTA, 1mM DTT, and 50% glycerol.

Stability

For short-term storage (up to 4 weeks), keep at 4°C. For extended periods, store frozen at -20°C. Adding a carrier protein like 0.1% HSA or BSA is recommended for long-term storage. Minimize repeated freeze-thaw cycles.

Reaction Conditions

Optimal reaction conditions are: 100mM KCl, 50mM Tris-HCl (pH 8.5), 20mM MgCl2, 0.1mM EDTA, 1mM DTT, 2% glycerol, at a temperature of 65°C.

Purity

The purity is greater than 95% as determined by SDS-PAGE analysis.

Preparation Protocol

1. Perform initial PCR amplification and purify the resulting fragments using the Qiagen QIAquick PCR purification kit. Elute the purified DNA in distilled water. 2. Dilute the purified PCR product to a concentration of 250 ng/µl in a buffer containing 10 mM Tris–HCl (pH 7.8) and 50 mM NaCl. Heat the solution to 95°C for 5 minutes, followed by a gradual cooling to 25°C at a rate of 0.1°C per second. 3. Prepare a binding buffer containing 20 mM Tris–HCl (pH 7.8), 10 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 5% glycerol. Combine the annealed PCR product with the binding buffer, adjusting the final DNA concentration to 11.5 ng/µl. Introduce MutS dimers to a final concentration of 950 nM. 4. Incubate the mixture at room temperature for 10 minutes. Subsequently, add an equivalent volume of Ni-NTA beads pre-equilibrated with the binding buffer. Continue incubation at room temperature for an additional 30 minutes. 5. Carefully separate the beads by centrifugation. Collect the supernatant for further applications, such as a second round of PCR or cloning.

Applications

This product is recommended for the following applications: 1. Removing mismatched DNA from gene synthesis reactions (error correction). 2. Detection and removal of mutations in DNA sequences. 3. Rapid isothermal detection of single nucleotide polymorphisms (SNPs).

Synonyms

MutS, Thermus Aquaticus DNA Mismatch Repair Protein, DNA mismatch repair protein MutS.

Source

Escherichia Coli.

Amino Acid Sequence

MEGMLKGEGPGPLPPLLQQYVELRDQYPDYLLLFQVGDFYECFGEDAERLARALG

LVLTHKTSKDFTTPMAGIPLRAFEAYAERLLKMGFRLAVADQVEPAEEAEGLVRREV

TQLLTPGTLLQESLLPREANYLAAIATGDGWGLAFLDVSTGEFKGTVLKSKSALYDELF

RHRPAEVLLAPELLENGAFLDEFRKRFPVMLSEAPFEPEGEGPLALRRARGALLAYAQ

RTQGGALSLQPFRFYDPGAFMRLPEATLRALEVFEPLRGQDTLFSVLDETRTAPGRRL

LQSWLRHPLLDRGPLEARLDRVEGFVREGALREGVRRLLYRLADLERLATRLELGRASP

KDLGALRRSLQILPELRALLGEEVGLPDLSPLKEELEAALVEDPPLKVSEGGLIREGYDPD

LDALRAAHREGVAYFLELEERERERTGIPTLKVGYNAVFGYYLEVTRPYYERVPKEYRPV

QTLKDRQRYTLPEMKEKEREVYRLEALIRRREEEVFLEVRERAKRQAEALREAARILAEL

DVYAALAEVAVRYGYVRPRFGDRLQIRAGRHPVVERRTEFVPNDLEMAHELVLITGPN

MAGKSTFLRQTALIALLAQVGSFVPAEEAHLPLFDGIYTRIGASDDLAGGKSTFMVEM

EEVALILKEATENSLVLLDEVGRGTSSLDGVAIATAVAEALHERRAYTLFATHYFELTAL

GLPRLKNLHVAAREEAGGLVFYHQVLPGPASKSYGVEVAAMAGLPKEVVARARALLQ

Product Science Overview

Introduction

The DNA mismatch repair (MMR) system is a critical mechanism that maintains the fidelity of DNA replication by correcting errors that escape the proofreading activity of DNA polymerases. One of the key proteins involved in this process is MutS, which recognizes and binds to mismatched bases in DNA. The Thermus aquaticus DNA mismatch repair protein MutS recombinant is a thermostable variant of this protein, produced through recombinant DNA technology.

Origin and Significance

Thermus aquaticus is a thermophilic bacterium that thrives in high-temperature environments, such as hot springs. The MutS protein from Thermus aquaticus is particularly valuable due to its thermostability, which allows it to function effectively at elevated temperatures. This property makes it an excellent candidate for various biotechnological applications, including PCR (polymerase chain reaction) and other DNA manipulation techniques that require high temperatures.

Structure and Function

The MutS protein is a member of the ABC ATPase superfamily and functions as a homodimer. Each subunit of the dimer contains several domains responsible for DNA binding, ATPase activity, and interaction with other proteins involved in the MMR pathway. The protein recognizes and binds to mismatched bases in duplex DNA, inducing a sharp kink in the DNA at the site of the mismatch. This binding event triggers a series of downstream processes that ultimately lead to the repair of the mismatch.

Recombinant Production

The recombinant form of Thermus aquaticus MutS is produced in Escherichia coli. The gene encoding the MutS protein is cloned into an expression vector, which is then introduced into E. coli cells. The bacteria are cultured under conditions that induce the expression of the MutS protein. The protein is subsequently purified using chromatographic techniques, often involving a His-tag for affinity purification. The resulting recombinant protein retains the thermostability and functional properties of the native protein.

Applications

The thermostable nature of Thermus aquaticus MutS makes it particularly useful in high-temperature applications. It is commonly used in PCR to enhance the fidelity of DNA amplification by correcting mismatches that occur during the replication process. Additionally, it is employed in various DNA manipulation techniques, such as cloning and sequencing, where high temperatures are required to denature DNA.

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