PURIFICATION AND CHARACTERIZATION OF TWO MEMBERS OF THE PROTEIN TYROSINE PHOSPHATASE FAMILY: DUAL SPECIFICITY PHOSPHATASE PVP AND LOW MOLECULAR WEIGHT PHOSPHATASE WZB By Paula A. Livingston A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Science in Partial Fulfillment for the Degree of Master of Science Florida Atlantic University Boca Raton, Florida December 2009 i ACKNOWLEDGMENTS The author wishes to thank her thesis advisor, Dr. Stefan Vetter, and the members of her committee, Dr. Estelle Leclerc and Dr. Predrag Cudic, for their advice and support throughout her years at Florida Atlantic University. The author also wishes to thank her family for their unending support and encouragement without which this would not have been possible. The author would also like to thank her nephew and niece, Griffin and Skye, for inspiring her throughout her entire graduate experience. iii ABSTRACT Author: Paula A. Livingston Title: Purification and Characterization of Two Members of the Protein Tyrosine Phosphatase Family: Dual Specificity Phosphatase PVP and Low Molecular Weight Phosphatase WZB Institution: Florida Atlantic University Thesis Advisor: Dr. Stefan W. Vetter Degree: Master of Science Year: 2009 Two protein tyrosine phosphatases, dual specificity phosphatase PVP and low molecular weight phosphatase WZB were purified and characterized. PVP was expressed as inclusion bodies and a suitable purification and refolding method was devised. Enzyme kinetics revealed that p-nitrophenylphosphate and β-naphthyl phosphate were substrates with KM of 4.0mM and 8.1mM respectively. PVP showed no reactivity towards phosphoserine. Kinetic characterization of WZB showed that only p- nitrophenylphosphate was a substrate with no affinity for β-naphthyl phosphate and phosphoserine. Optimal conditions for activity with PNPP were found at a pH of 5 with -1 -1 -1 a KM of 1.1mM, kcat of 35.4s and kcat/KM of 32.2s mM . Inhibition studies showed that phosphate, fluoride, and molybdate were competitive inhibitors with Ki of 3.2mM, 71.7mM, and 50.4μM respectively and hydrogen peroxide abolished activity. Active site mutants of WZB Cys9Ser and Asp115Asn showed no activity. iv PURIFICATION AND CHARACTERIZATION OF TWO MEMBERS OF THE PROTEIN TYROSINE PHOSPHATASE FAMILY: DUAL SPECIFICITY PHOSPHATASE PVP AND LOW MOLECULAR WEIGHT PHOSPHATASE WZB List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1 – Introduction ......................................................................................................1 1.1 – The protein phosphatase superfamily .............................................................1 PPP family ......................................................................................................2 PPM family .....................................................................................................2 PTP family ......................................................................................................3 Aspartate based family ....................................................................................4 1.2 – Overview of protein tyrosine phosphatases ....................................................4 1.3 – Classification of PTPs .....................................................................................5 Phosphotyrosine specific PTPs .......................................................................6 Dual specificity phosphatases .........................................................................7 Cdc25 phosphatases ........................................................................................9 Low molecular weight phosphatases ............................................................10 1.4 – Biological functions of PTPs ........................................................................11 PTPs and cancer ............................................................................................11 PTPs and Alzheimer’s...................................................................................13 v PTPs and diabetes .........................................................................................14 1.5 – Molecular structure of PTPs .........................................................................17 1.6 – Mechanism of catalysis of PTPs ...................................................................20 1.7 – PTPs in eukaryotes and prokaryotes .............................................................23 1.8 – Poxvirus phosphatases ..................................................................................26 1.9 – Low molecular weight phosphatase WZB in Escherichia coli .....................27 Chapter 2 – Materials and Methods ...................................................................................29 2.1 – Instrumentation and Buffers .........................................................................29 2.2 – Transformation of plasmid into expression hosts .........................................30 2.3 – Cell growth and expression of phosphatases ................................................32 2.4 – Isolation of inclusion bodies .........................................................................36 2.5 – Nickel affinity chromatography ....................................................................37 2.6 – Ion exchange chromatography ......................................................................38 2.7 – Gel filtration chromatography.......................................................................38 2.8 – Refolding matrix design ................................................................................39 2.9 – Refolding protocol ........................................................................................40 2.10 – Enzyme kinetics ..........................................................................................41 2.11 – Fitting of enzyme kinetic data.....................................................................42 2.12 – Synthesis of non-detergent sulphobetaines (NDSBs) .................................43 2.13 – Molecular cloning of WZB mutants ...........................................................45 Chapter 3 – Results: cell growth, expression, purification and kinetic characterization of PVP ....................................................................................................................46 3.1 – Growth and expression of PVP .....................................................................46 vi 3.2 – Purification of PVP .......................................................................................48 3.3 – Refolding of His6-tagged PVP .....................................................................51 3.4 – Enzyme kinetics of PVP ...............................................................................56 Chapter 4 – Results: cloning, cell growth, expression, purification and kinetic characterization of WZB ........................................................................................60 4.1 – Growth and expression of WZB ...................................................................60 4.2 – Purification of WZB .....................................................................................61 4.3 – Enzyme kinetics of WZB ..............................................................................63 4.4 – Inhibitory effects on WZB ............................................................................68 4.5 – Comparison of enzymatic activity between the two forms of WZB ............76 4.6 – Enzyme activity of WZB mutants.................................................................78 Chapter 5 – Summary ........................................................................................................80 References ..........................................................................................................................83 vii LIST OF TABLES Table 1 – Cdc25 overexpression found in different cancers..............................................13 Table 2 – Prokaryotic PTPs ...............................................................................................24 Table 3 – DNA and amino acid sequence of both forms of PVP ......................................35 Table 4 – DNA and amino acid sequence of WZB ...........................................................36 Table 5 – Initial refolding buffer systems for His6-tagged PVP .......................................39 Table 6 – Additional refolding buffer systems for His6-tagged PVP ................................40 Table 7 – Enzymatic results of PVP with various substrates .............................................56 Table 8 – Results of inhibition studies with WZB .............................................................69 Table 9 – Comparison of WZB activity between two forms of WZB ...............................76 viii LIST OF FIGURES Figure 1 – Protein phosphorylation as regulated by kinases and phosphatases ...................1 Figure 2 – Catalytic mechanism of PP1 (member of PPP family) ......................................3 Figure 3 – Classification of the PTP superfamily ................................................................7 Figure 4 – Cdc25 phosphatases and cyclin-dependent kinases .........................................10 Figure