Mapping The Intrinsic Carbonyl Reducing Activity of Thioredoxin Reductase

Mapping the Intrinsic Carbonyl Reducing Activity of Thioredoxin ReductasePhuong PhamDr. Gary MerrillSummer 20111InterestsExplore the functions of thioredoxin reductaseOnly known enzyme to reduce thioredoxinRecent research suggested roles in reducing toxic carbonyl-containing compounds in cells

http://www.molecularstructure.org/entry.php?pdb=1ZKQThioredoxin (Trx)Small protein (104 amino acids)Supplies electrons for enzymatic and regulatory reactionsReduced again by thioredoxin reductase

http://en.engormix.com/MA-dairy-cattle/articles/selenium-metabolism-animals-relationship-t363/p0.htmThioredoxin 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

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.htmHypothesisOnly site 1 of thioredoxin reductase is needed for carbonyl reductionThe selenocysteine-containing site 2 is unnecessary

Problems with BacteriaIn eukaryotes, the amino acid selenocysteine (Sec) is incorporated opposite UGA codonBecause bacteria cannot insert Sec opposite UGA, site 2 is inactive Recombinant Txnrd1 cannot reduce thioredoxin

http://www.edvotek.com/300Previous StudiesTo obtain active recombinant Txnrd1 protein, the Sec codon is changed to a cysteine (Cys) codonThe 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

Site 2 might not be necessary in reducing carbonyls

MetXXXGlyCysSecGlyMetXXXGlyCysCysGlyMetXXXGlyCysSTOPMouse Txnrd1 SequencesWild Type Txnrd1 sequence expressed in eukaryotic cellsEngineered Txnrd1 sequence expressed in E. coliWild Type Txnrd1 sequence expressed in E. coliReduces thioredoxin and carbonylsLower level of thiorexin reduction; still reduces carbonylsRelated Interest: Glutathione ReductaseReduces GlutathioneSimilar dimer structure to Txnrd1Participates in DNA synthesisDefends against oxidative stresshttp://www.asiaandro.com/archive/1008-682X/5/231.htm

ObjectivesCompare TR carbonyl reduction of mammals against yeast & bacteriaCompare TR carbonyl reduction ability against GRCarbonyl reduction is specific to TR in mammals?Reduction ofMouse TRYeast TRYeast GRE. coli TRE. coli GRTrx N/AN/AGrxN/AN/AN/ACarbonyl????ProcedureGenerated mTR1 insert through PCR reactionInserted into TOPO cloning vector and transformed with E. coliPerformed PCR to determine orientation of mTR1 insertCut insert out with Nde1 and BamH1 restriction enzymesInserted into pET28a expression vector and transformed with E. coliDigested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coliHarvested enzyme using TALON Metal Affinity Resins

PCR Product mTR1 Insert

Added Nde1 restriction site at beginning of the insertVerified presence of insert on electrophoresis gel Approx. 1500 base pairs 200030001000~1500 bpProcedureGenerated mTR1 insert through PCR reactionInserted into TOPO cloning vector and transformed with E. coliPerformed PCR to determine orientation of mTR1 insertCut insert out with Nde1 and BamH1 restriction enzymesInserted into pET28a expression vector and transformed with E. coliDigested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coliHarvested enzyme using TALON Metal Affinity Resins

Insert into Vector

http://www.nmr.chem.uu.nl/users/rob/efc.htmlCloning Vector - TOPOBefore expressing to obtain more genesTaq polymerase adds single A to inserts 3 endsTransformed with Top10 Competent E. colihttp://www.clas.ufl.edu/jur/200209/papers/paper_henry.htmlmTR1 insertAA

ProcedureGenerated mTR1 insert through PCR reactionInserted into TOPO cloning vector and transformed with E. coliPerformed PCR to determine orientation of mTR1 insertCut insert out with Nde1 and BamH1 restriction enzymesInserted into pET28a expression vector and transformed with E. coliDigested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coliHarvested enzyme using TALON Metal Affinity Resins

Determined Orientation of InsertTOPO Vector3.9 kbmTR1 insert ~1500bpTOPO Vector3.9 kbmTR1 insert~1500bppTOPO-mTR1 pTOPO-mTR1 AUGf AUGf TGArTGAr m13r m13f m13r m13f pLacpLacNde1Nde1Determined Orientation of InsertOrientationPrimersPredicted PCR ProductTGAr + m13r1500 bpTGAr + m13fNo productTGAr + m13rNo productTGAr + m13f1500 bpDetermined Orientation of Insert

Insert is in the reverse orientation in plasmidChose the 7th and 9th clonesNote: Abnormality in all clones have same orientation1000100020002000~1500 bp712436589No Product712436589TGAr + m13fTGAr + m13rProcedureGenerated mTR1 insert through PCR reactionInserted into TOPO cloning vector and transformed with E. coliPerformed PCR to determine orientation of mTR1 insertCut insert out with Nde1 and BamH1 restriction enzymesInserted into pET28a expression vector and transformed with E. coliDigested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coliHarvested enzyme using TALON Metal Affinity Resins

Clones DigestionUsed Nde1 and BamH1 restriction enzymes to cut out insert BamH1 2531496TOPO Vector3.9 kbmTR1 insert~1500bpAUGf TGAr m13f m13r pLacNde1EcoR5 314ProcedureGenerated mTR1 insert through PCR reactionInserted into TOPO cloning vector and transformed with E. coliPerformed PCR to determine orientation of mTR1 insertCut insert out with Nde1 and BamH1 restriction enzymesInserted into pET28a expression vector and transformed with E. coliDigested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coliHarvested enzyme using TALON Metal Affinity Resins

Expression Vector pET28aReadied insert for expressionUsed pET28a already cut at Nde1 and BamH1 sitesTransformed with DH5 competent E. colihttp://www.genomex.com/vector_maps/pET28_map.pdf

pET28a5.4 kbProcedureGenerated mTR1 insert through PCR reactionInserted into TOPO cloning vector and transformed with E. coliPerformed PCR to determine orientation of mTR1 insertCut insert out with Nde1 and BamH1 restriction enzymesInserted into pET28a expression vector and transformed with E. coliDigested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coliHarvested enzyme using TALON Metal Affinity Resins

Digestion with Nde1 and BamH1Clones 7 and 16 weakly showed ~1500 bp insertProceeded with transformation into BL21 E. coliAlso used clone 5 as control

100010002000200091011121314151612345678

25015010075503725201510kDProtein Gel5 71657 16UninducedIPTG Induced 53 kD for Txnrd1according to literatureProcedureGenerated mTR1 insert through PCR reactionInserted into TOPO cloning vector and transformed with E. coliPerformed PCR to determine orientation of mTR1 insertCut insert out with Nde1 and BamH1 restriction enzymesInserted into pET28a expression vector and transformed with E. coliDigested with restriction enzymes to verified presence of vector and insert and transformed into BL21 Competent E. coliHarvested enzyme using TALON Metal Affinity Resins

TALON Resin HarvestUsed the TALON resin beads with cobalt to bind to polyhistidine tag on proteinsUnsuccessful in binding protein to resin

Conclusion: Proteins might be insoluble or not able to bind to resinshttp://www.clontech.com/US/Support/Applications/His-Tagged_Protein_Purification/Ni-NTA_Resin_vs._Talon?sitex=10020:22372:USRepeat with Different Truncation of Trxnd1MetXXXGlySerSerGlyWild Type Txnrd1 sequence expressed in eukaryotic cellsMetXXXGlyCysSecGlyRe-engineered Txnrd1 sequence expressed in E. coliReplace Cys and Sec with Serine (Ser)Future WorksContinue to express newly truncated gene and purify the proteinCan observe cellular activity through microscopy of fluorescent staining and morphologyIdentify the mechanism of carbonyl reduction in thioredoxin reductase

AcknowledgementDr. Gary MerrillDr. Kevin AhernFrancis Cripps FoundationEnvironmental Health Sciences CenterHoward Hughes Medical Institute