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Mapping the Intrinsic Carbonyl Reducing Activity of Thioredoxin Reductase

Mapping the Intrinsic Carbonyl Reducing Activity of Thioredoxin Reductase. Phuong Pham Dr. Gary Merrill Summer 2011. Interests. http://www.molecularstructure.org/entry.php?pdb=1ZKQ. Explore the functions of thioredoxin reductase Only known enzyme to reduce thioredoxin

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Mapping the Intrinsic Carbonyl Reducing Activity of Thioredoxin Reductase

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  1. Mapping the Intrinsic Carbonyl Reducing Activity of Thioredoxin Reductase Phuong Pham Dr. Gary Merrill Summer 2011

  2. Interests http://www.molecularstructure.org/entry.php?pdb=1ZKQ Explore the functions of thioredoxin reductase Only known enzyme to reduce thioredoxin Recent research suggested roles in reducing toxic carbonyl-containing compounds in cells

  3. Thioredoxin (Trx) • Small protein (104 amino acids) • Supplies electrons for enzymatic and regulatory reactions • Reduced again by thioredoxin reductase http://en.engormix.com/MA-dairy-cattle/articles/selenium-metabolism-animals-relationship-t363/p0.htm

  4. Thioredoxin Reductase (Txnrd1) • Recent studies suggests it also reduces carbonyls (ketones and aldehydes) • Two active sites (site 1 near N terminus; site 2 near C terminus) • Site 2 has the unusual amino acid selenocysteine http://www.asiaandro.com/archive/1008-682X/5/231.htm

  5. Thioredoxin Reductase (Cont.) • Mechanism of thioredoxin reduction: NADPH FAD  Site 1 Site 2 Trx • Mechanism of carbonyl reduction is unknown http://www.asiaandro.com/archive/1008-682X/5/231.htm

  6. Hypothesis Only site 1 of thioredoxin reductase is needed for carbonyl reduction The selenocysteine-containing site 2 is unnecessary

  7. Problems with Bacteria http://www.edvotek.com/300 In eukaryotes, the amino acid selenocysteine (Sec) is incorporated opposite UGA codon Because bacteria cannot insert Sec opposite UGA, site 2 is inactive Recombinant Txnrd1 cannot reduce thioredoxin

  8. Previous Studies  Site 2 might not be necessary in reducing carbonyls To obtain active recombinant Txnrd1 protein, the Sec codon is changed to a cysteine (Cys) codon The Cys form of Txnrd1 is 10 times less active than wild type form in reducing thioredoxin However, this form is fully active in reducing the carbonyl-containing compound menadione

  9. Mouse Txnrd1 Sequences Met X X……… X GlyCys Sec Gly Reduces thioredoxin and carbonyls • Wild Type Txnrd1 sequence expressed in E. coli Met X X……… X GlyCysCysGly Lower level of thiorexin reduction; still reduces carbonyls Met X X……… X GlyCys STOP Wild Type Txnrd1 sequence expressed in eukaryotic cells Engineered Txnrd1 sequence expressed in E. coli

  10. Related Interest: Glutathione Reductase • Reduces Glutathione • Similar dimer structure to Txnrd1 • Participates in DNA synthesis • Defends against oxidative stress http://www.asiaandro.com/archive/1008-682X/5/231.htm

  11. Objectives Compare TR carbonyl reduction of mammals against yeast & bacteria Compare TR carbonyl reduction ability against GR Carbonyl reduction is specific to TR in mammals?

  12. Procedure Generated mTR1 insert through PCR reaction Inserted into TOPO cloning vector and transformed with E. coli Performed PCR to determine orientation of mTR1 insert Cut insert out with Nde1 and BamH1 restriction enzymes Inserted into pET28a expression vector and transformed with E. coli Digested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coli Harvested enzyme using TALON Metal Affinity Resins

  13. PCR Product – mTR1 Insert • Added Nde1 restriction site at beginning of the insert • Verified presence of insert on electrophoresis gel • Approx. 1500 base pairs 3000 2000 ~1500 bp 1000

  14. Procedure Generated mTR1 insert through PCR reaction Inserted into TOPO cloning vector and transformed with E. coli Performed PCR to determine orientation of mTR1 insert Cut insert out with Nde1 and BamH1 restriction enzymes Inserted into pET28a expression vector and transformed with E. coli Digested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coli Harvested enzyme using TALON Metal Affinity Resins

  15. Insert into Vector http://www.nmr.chem.uu.nl/users/rob/efc.html

  16. Cloning Vector - TOPO mTR1 insert A • Before expressing to obtain more genes • Taq polymerase adds single A to insert’s 3’ ends • Transformed with Top10 Competent E. coli A http://www.clas.ufl.edu/jur/200209/papers/paper_henry.html

  17. Procedure Generated mTR1 insert through PCR reaction Inserted into TOPO cloning vector and transformed with E. coli Performed PCR to determine orientation of mTR1 insert Cut insert out with Nde1 and BamH1 restriction enzymes Inserted into pET28a expression vector and transformed with E. coli Digested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coli Harvested enzyme using TALON Metal Affinity Resins

  18. Determined Orientation of Insert pTOPO-mTR1  AUGf m13r  m13f  pLac mTR1 insert ~1500bp TOPO Vector3.9 kb  TGAr Nde1 pTOPO-mTR1  AUGf m13r  m13f  pLac mTR1 insert ~1500bp TOPO Vector 3.9 kb TGAr  Nde1

  19. Determined Orientation of Insert

  20. Determined Orientation of Insert • Insert is in the reverse orientation in plasmid • Chose the 7th and 9th clones • Note: Abnormality in all clones have same orientation 1 2 3 6 7 8 9 4 5 2000 1000 TGAr + m13f ~1500 bp 1 3 4 5 6 7 8 9 2 2000 No Product 1000 TGAr + m13r

  21. Procedure Generated mTR1 insert through PCR reaction Inserted into TOPO cloning vector and transformed with E. coli Performed PCR to determine orientation of mTR1 insert Cut insert out with Nde1 and BamH1 restriction enzymes Inserted into pET28a expression vector and transformed with E. coli Digested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coli Harvested enzyme using TALON Metal Affinity Resins

  22. Clones Digestion Used Nde1 and BamH1 restriction enzymes to cut out insert 1496 AUGf m13r  m13f  pLac mTR1 insert ~1500bp TOPO Vector 3.9 kb TGAr  BamH1 253 EcoR5 314 Nde1

  23. Procedure Generated mTR1 insert through PCR reaction Inserted into TOPO cloning vector and transformed with E. coli Performed PCR to determine orientation of mTR1 insert Cut insert out with Nde1 and BamH1 restriction enzymes Inserted into pET28a expression vector and transformed with E. coli Digested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coli Harvested enzyme using TALON Metal Affinity Resins

  24. Expression Vector – pET28a • Readied insert for expression • Used pET28a already cut at Nde1 and BamH1 sites • Transformed with DH5αcompetent E. coli pET28a 5.4 kb http://www.genomex.com/vector_maps/pET28_map.pdf

  25. Procedure Generated mTR1 insert through PCR reaction Inserted into TOPO cloning vector and transformed with E. coli Performed PCR to determine orientation of mTR1 insert Cut insert out with Nde1 and BamH1 restriction enzymes Inserted into pET28a expression vector and transformed with E. coli Digested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coli Harvested enzyme using TALON Metal Affinity Resins

  26. Digestion with Nde1 and BamH1 2000 1000 2000 1000 Clones 7 and 16 weakly showed ~1500 bp insert Proceeded with transformation into BL21 E. coli Also used clone 5 as control

  27. Protein Gel Uninduced IPTG Induced kD 53 kD for Txnrd1 according to literature

  28. Procedure Generated mTR1 insert through PCR reaction Inserted into TOPO cloning vector and transformed with E. coli Performed PCR to determine orientation of mTR1 insert Cut insert out with Nde1 and BamH1 restriction enzymes Inserted into pET28a expression vector and transformed with E. coli Digested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coli Harvested enzyme using TALON Metal Affinity Resins

  29. TALON Resin Harvest Conclusion: Proteins might be insoluble or not able to bind to resins http://www.clontech.com/US/Support/Applications/His-Tagged_Protein_Purification/Ni-NTA_Resin_vs._Talon?sitex=10020:22372:US Used the TALON resin beads with cobalt to bind to polyhistidine tag on proteins Unsuccessful in binding protein to resin

  30. Repeat with Different Truncation of Trxnd1 Wild Type Txnrd1 sequence expressed in eukaryotic cells Met X X……… X GlyCys Sec Gly Re-engineered Txnrd1 sequence expressed in E. coli Met X X……… X Gly Ser SerGly Replace Cys and Sec with Serine (Ser)

  31. Future Works Continue to express newly truncated gene and purify the protein Can observe cellular activity through microscopy of fluorescent staining and morphology Identify the mechanism of carbonyl reduction in thioredoxin reductase

  32. Acknowledgement Dr. Gary Merrill Dr. Kevin Ahern Francis Cripps Foundation Environmental Health Sciences Center Howard Hughes Medical Institute

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