PMID- 12767817
OWN - NLM
STAT- MEDLINE
DCOM- 20040317
LR  - 20191107
IS  - 1046-5928 (Print)
IS  - 1046-5928 (Linking)
VI  - 29
IP  - 2
DP  - 2003 Jun
TI  - Overexpression and purification of isotopically labeled Escherichia coli MutH for 
      NMR studies.
PG  - 252-8
AB  - MutH is one of the enzymes involved in the methyl directed -GATC-based DNA repair 
      system. We report a significantly optimized protocol to prepare isotopically (15N 
      and/or 13C) labeled MutH in minimal medium with high yields for NMR studies. 
      Under the various conditions that we have standardized for the affinity 
      purification of His(6) MutH, the yield of the purified MutH has been estimated to 
      be 35-40 mg of protein from 1liter of M9 minimal media. The yield, thus, obtained 
      by this method is significantly higher than those of previously reported methods. 
      Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass 
      spectroscopy analysis revealed that the protein was pure and existed essentially 
      in a monomeric form. Uniformly 15N-labeled protein, thus, produced has been 
      characterized by recording a sensitivity enhanced 2D [15N]-[1H] HSQC spectrum. 
      The dispersion seen in 15N-1H cross-peaks indicates the presence of a 
      well-ordered structure for MutH and proper folding of the purified protein. The 
      spectrum confirms further the existence of MutH as a monomer.
FAU - Dutta, Arnob
AU  - Dutta A
AD  - Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi 
      Bhabha Road, Colaba, Mumbai 400 005, India.
FAU - Rao, Basuthkar J
AU  - Rao BJ
FAU - Chary, Kandala V R
AU  - Chary KV
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Carbon Isotopes)
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (Escherichia coli Proteins)
RN  - 0 (Nitrogen Isotopes)
RN  - 0 (Recombinant Proteins)
RN  - EC 3.1.- (Endodeoxyribonucleases)
RN  - EC 3.1.21.- (methyl-directed mismatch repair protein, E coli)
RN  - EC 6.5.1.- (DNA Repair Enzymes)
SB  - IM
MH  - Amino Acid Sequence
MH  - Carbon Isotopes
MH  - Chromatography, Affinity
MH  - DNA Repair
MH  - *DNA Repair Enzymes
MH  - DNA-Binding Proteins/*biosynthesis/*chemistry/isolation & purification
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Endodeoxyribonucleases/*biosynthesis/*chemistry/isolation & purification
MH  - Escherichia coli/*enzymology/genetics/metabolism
MH  - Escherichia coli Proteins
MH  - Isotope Labeling/methods
MH  - Molecular Sequence Data
MH  - Nitrogen Isotopes
MH  - Nuclear Magnetic Resonance, Biomolecular/methods
MH  - Protein Conformation
MH  - Recombinant Proteins/biosynthesis/chemistry/isolation & purification
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
EDAT- 2003/05/28 05:00
MHDA- 2004/03/18 05:00
CRDT- 2003/05/28 05:00
PHST- 2003/05/28 05:00 [pubmed]
PHST- 2004/03/18 05:00 [medline]
PHST- 2003/05/28 05:00 [entrez]
AID - S1046592803000561 [pii]
AID - 10.1016/s1046-5928(03)00056-1 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2003 Jun;29(2):252-8. doi: 10.1016/s1046-5928(03)00056-1.