TOPO I (Human DNA Topoisomerase I, Core Domain 197-651)

• Species; Human
• Expression Host: Baculovirus-insect cell
• Tag: His-tag
• Purity: 90%
• Molecular Weight: 55.9 kDa.
• Gene Accession Number: NM_003286.

Purification and Quality Control

The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns.
The purified TOPO I (Core Domain 197-651) is greater than 95% homogeneous based on SDS-PAGE analysis.

Unit Definition (Activity)

1 unit equals 1 nanogram purified protein. 1 – 10 units (ng) are sufficient for in vitro reconstituted relaxation assay in a 20μl reaction and contain no detectable DNA relaxation activity by itself.
Variable exist in different lots.

Applications

Purified core domain of topo I can be used for DNA binding and protein-protein interaction assays. It can be also reconstituted with the C-terminal domain to restore the DNA relaxation activity in vitro.

Formulation and Storage

The protein is in 20mM Tris-HCl pH7.9,100mM NaCl, 0.2mM EDTA, 1mM DTT and 20% glycerol. Stored at -70°C before use. Avoid repeated freeze thaw cycles.

Protein Sequence

KEEEQKWKWW EEERYPEGIK WKFLEHKGPV FAPPYEPLPE NVKFYYDGKV MKLSPKAEEV ATFFAKMLDH EYTTKEIFRK NFFKDWRKEM TNEEKNIITN LSKCDFTQMS QYFKAQTEAR KQMSKEEKLK IKEENEKLLK EYGFCIMDNH KERIANFKIE PPGLFRGRGN HPKMGMLKRR IMPEDIIINC SKDAKVPSPP PGHKWKEVRH DNKVTWLVSW TENIQGSIKY IMLNPSSRIK GEKDWQKYET ARRLKKCVDK IRNQYREDWK SKEMKVRQRA VALYFIDKLA LRAGNEKEEG ETADTVGCCS LRVEHINLHP ELDGQEYVVE FDFLGKDSIR YYNKVPVEKR VFKNLQLFME NKQPEDDLFD RLNTGILNKH LQDLMEGLTA KVFRTYNASI TLQQQLKELT APDENIPAKI LSYNRANRAV AILCNHQRAP PKTFEKSMMN LQTKI

Background

Human DNA topoisomerase I is the best studied of the DNA topoisomerase family. It catalyzes the relaxation of both positive and negative supercoils without the requirement of energy. In addition to DNA replication and transcriptional activation, DNA topoisomerase I also plays a major role in pre-mRNA splicing, cell cycle and other gene regulatory pathways during cell growth and development (1). The core domain spanning from amino acids 215 to 636 is highly conserved and retains DNA binding activity (2,3). Topo I has been found to nick the DNA with a preference of 5’-(A/T)(G/C)(A/T)T-3’ (4,5). Camptothecin and its analogs have been tested as potent anticancer compounds by stabilizing topo I-DNA complex, thereby inhibiting both DNA and RNA synthesis (6-9).

References

1. Champoux, J.J. (2001) Annu. Rev. Biochem. 70, 369-413
2. Stewart, L. et al., (1996) J. Biol. Chem. 271, 7593-7601
3. Redinbo, M.R. et al., (1998) Science 279, 1504-1513
4. Tanizawa, A. et al., (1993) Nucleic Acids Res. 21, 5157-5166
5. Bouven, B.J. et al., (1985) Cell 41, 541-551
6. Hsiang, Y.H. et al., (1985) J. Biol. Chem. 260, 14873-14878
7. Bjornsti, M.A. et al., (1989) Cancer Res. 49, 6318-6323
8. Jaxel, C. et al., (1989) Cancer Res. 49, 1465-1469
9. Pommier, Y. et al., (1995) Proc. Natl. Acad. Sci. USA 92, 8861-88655

DISCLAIMER

This products is recommended For RESEARCH USE ONLY and is Not qualified for Use in Diagnostic or Therapeutic Procedures.