Phuong Pham Dr. Gary Merrill Summer 2011

 Explore the functions of thioredoxin reductase  Only known enzyme to reduce thioredoxin  Recent research suggested roles in reducing toxic carbonyl-containing compounds in cells

 Small protein (104 amino acids)  Supplies electrons for enzymatic and regulatory reactions  Reduced again by thioredoxin reductase

 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

 Mechanism of thioredoxin reduction: NADPH  FAD  Site 1  Site 2  Trx  Mechanism of carbonyl reduction is unknown

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

 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

 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  Site 2 might not be necessary in reducing carbonyls

MetXX………XGlyCysSecGly MetXX………XGlyCysCysGly MetXX………XGlyCysSTOP Wild Type Txnrd1 sequence expressed in eukaryotic cells Engineered Txnrd1 sequence expressed in E. coli Wild Type Txnrd1 sequence expressed in E. coli Reduces thioredoxin and carbonyls Lower level of thiorexin reduction; still reduces carbonyls

 Reduces Glutathione  Similar dimer structure to Txnrd1  Participates in DNA synthesis  Defends against oxidative stress

 Compare TR carbonyl reduction of mammals against yeast & bacteria  Compare TR carbonyl reduction ability against GR  Carbonyl reduction is specific to TR in mammals? Reduction ofMouse TRYeast TRYeast GRE. coli TRE. coli GR Trx N/A N/A Grx N/A N/A Carbonyl ????

 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

 Added Nde1 restriction site at beginning of the insert  Verified presence of insert on electrophoresis gel  Approx base pairs ~1500 bp

 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

 Before expressing to obtain more genes  Taq polymerase adds single A to insert’s 3’ ends  Transformed with Top10 Competent E. coli mTR1 insert A A

 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

TOPO Vector 3.9 kb mTR1 insert ~1500bp TOPO Vector 3.9 kb mTR1 insert~1500bp pTOPO-mTR1  pTOPO-mTR1  AUGf   AUGf  TGAr TGAr  m13r  m13f  m13r  m13f  pLac Nde1

OrientationPrimers Predicted PCR Product  TGAr + m13r1500 bp  TGAr + m13fNo product  TGAr + m13rNo product  TGAr + m13f1500 bp

 Insert is in the reverse orientation in plasmid  Chose the 7 th and 9 th clones  Note: Abnormality in all clones have same orientation ~1500 bp No Product TGAr + m13f TGAr + m13r

 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

Used Nde1 and BamH1 restriction enzymes to cut out insert BamH TOPO Vector 3.9 kb mTR1 insert~1500bp  AUGf TGAr  m13f  m13r  pLac Nde1 EcoR5 314

 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

 Readied insert for expression  Used pET28a already cut at Nde1 and BamH1 sites  Transformed with DH5 α competent E. coli pET28a 5.4 kb

 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

 Clones 7 and 16 weakly showed ~1500 bp insert  Proceeded with transformation into BL21 E. coli  Also used clone 5 as control

kD UninducedIPTG Induced 53 kD for Txnrd1 according to literature

 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

 Used the TALON resin beads with cobalt to bind to polyhistidine tag on proteins  Unsuccessful in binding protein to resin Conclusion: Proteins might be insoluble or not able to bind to resins

MetXX………XGlySerSerGly Wild Type Txnrd1 sequence expressed in eukaryotic cells MetXX………XGlyCysSecGly Re-engineered Txnrd1 sequence expressed in E. coli Replace Cys and Sec with Serine (Ser)

 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

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