Download - MBP-Putrescine Oxidase
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Putrescine Oxidase Vida Espinosa & Kathy WilsonBIOL 473 - Fall 2015
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Background• Putrescine Oxidase is also known as PuOx• Belongs to the MAO family of flavin-containing amine oxidases1
• MAO: structural family, monoamine oxidase• Contains one flavin adenine dinucleotide (FAD) per dimer
• FAD is a cofactor used in the catalysis of PuOx • Catalyzes oxidation of many different compounds, including
putrescine• Reducing the oxygen to hydrogen peroxide concurrent with the
previous stated oxidation1
• No coenzyme is needed for reaction, due to the electron acceptor being molecular oxygen
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Structure and Mechanism• Putrescine Oxidase exists as is a homodimer• Molecular Weight as homodimer: 198 kDa
• putrescine + O2 + H2O = 4-aminobutanal + NH3 + H2O2
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Protein Tags • GFP tag:
• Known as Green Fluorescent Protein• Emits a green fluorescent glow
• Allows for visualization of where and/or when the protein is expressed2
• Molecular weight: 27 kDa• MBP tag:
• Known as Maltose Binding Protein • New England Biolabs developed MBP in the 1980’s3
• Promotes stability of protein• Heightens solubility within protein • Molecular weight: 42 kDa
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Work Flow
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Transformation of GFP-PuOx• Transformation completed using T7
Express lysY Competent E. coli• Plasmid for the expression of GFP-PuOx
obtained from University of Kansas• Plasmid included GFP-PuOx gene• Also contained gene for ampicillin resistance
• Heat shock used to disrupt cell wall to allow introduction of the plasmid DNA
• After transformation, the cells were then streaked onto an ampicillin/LB agar plate, allowing only growth of the transformed cells.
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• Protein growth completed in 1X LB Broth, incubated overnight• Over-expression was completed after 3 hours and 30 minutes
adding 1M IPTG, incubated overnight• Cells were centrifuged
• Resuspended pellet was the crude sample of protein• Cells were lysed by sonification
• Resultant solution was the lysate same of protein
Protein Growth and Over-Expression of GFP-PuOx
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Q-Sepharose Anion Exchange Column of GFP-PuOx
• The Q-sepharose is a positively charged resin
• GFP-PuOx is a negatively charged molecule, so it will be attracted to the Q-sepharose and stick to the column when added
• Using a KCl concentration gradient, salting out occurred on the column and PuOx was eluted out
• PuOx interacted with the positive potassium ion • The negative chloride ion interacted with the positive resin
• Experiment completed in the cold room
Socratic
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GFP-PuOx After Purification• A280 completed on each of the
eluted fractions• Three peaks were found, each
peak distance was combined to create three separate samples
• Sample 1: tubes 4-11• Sample 2: tubes 12-18• Sample 3: tubes 19-24
• Samples were concentrated using 30,000 MWCO Amicon Filters
• About 250 L of each peak were 𝜇recovered
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Activity Assay of GFP-PuOx• Amplex Red Assay used to check for
activity• The generation of H2O2 is coupled to
the conversion of the Amplex Red reagent to fluorescent resorufin by HRP5.
• Of the three fractions, only one showed any activity
• Activity shown by development of bright pink color
• This assay was used for all subsequent activity assays and kinetics assays
Thermo Fisher Scientific
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Hydrogen Peroxide Standard Curve• A standard curve for hydrogen peroxide was built using
the Amplex Red Assay• Calculated the extinction coefficient to be 66, 615 M-1cm-1
• Literature value is 58,000 ± 5000 M-1cm-1
• This was later used to transform the kinetics data
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SDS-PAGE of GFP-PuOx• Gel electrophoresis showed that the fractions
were not well purified using the Q-sepharose column
• Possible reasons• Q-sepharose not regenerated properly• Q-sepharose stock was overused • Salting out not adequate• Q-sepharose was not this optimal method of purification
16 kDa17 kDa
28 kDa
38 kDa
49 kDa
62 kDa98 kDa
188 kDa
6 kDa
Fractions
13 2
4 kDa
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Histidine Tag Affinity Column of MBP-PuOx • Cell growth, overexpression, and lysis were
completed on cell stocks of MBP-PuOx as previously completed with GFP-PuOx
• Used 2X LB broth for overexpression• Nickel Column
• Histidine tag on PuOx has an affinity for the Nickel ion on the resin in the column• Imidazole has a higher affinity for Nickel ions than Histidine so will
knock off PuOx• Unbound proteins were washed from the column with a buffer containing a higher
concentration of Imidazole.• The concentration of Imidazole was increased again with elution
buffer to elute PuOx out of the column
Gentaur BVBA
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FAD Cofactor Content• Spectral Scan from 300 nm - 600 nm completed on purified protein
sample to determine FAD content • The absorbance of the sample at 450 nm was used to find the ratio of FAD to PuOx
• Ratio of FAD to PuOx in the sample was 1:1.68 ration• Ratio also expressed at .5945 FAD/PuOx monomer• The expected ratio is 1:2
• The ratio was later used to transform the kinetics data
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Enzyme Kinetics of MBP-PuOx
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Enzyme Kinetics of MBP-PuOx
Kcat Kcat/Km
Experimental 13.78s-1 211.93 s-1mM-1
Literature 20.7s-1 5900 s-1mM-1
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SDS-PAGE of MBP-PuOx• Gel electrophoresis showed a
heightening of purity through the stages of purification of the protein
• The stages of protein farther along the purification process had less bands than previous stages
• There was a noticeable jump of purification from the wash stage to the elutant stage
STD
Crude
Lysa
teLo
ading
Was
hEl
utan
t
16 kDa17 kDa
28 kDa
38 kDa
49 kDa
62 kDa
98 kDa
188 kDa
6 kDa
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Conclusion• Purified MBP-PuOx• Determined Kinetics of purified MBP-PuOx• Determined FAD cofactor Content of MBP-PuOx• Learned and improved different laboratory protocols
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Bibliography1. van Hellemond, E.W.; van Dijk, M.; Heuts, D.P.H.M.; Janssen, D.B; Fraaije, M.W. Discovery and
characterization of a putrescine oxidase from Rhodococcus erythropolis. Appl. Microbiol Biotechnol. (2008) 78:455-463.
2. Sanders, Jeremy K. M., and Sophie E. Jackson. "The Discovery and Development of the Green Fluorescent Protein, GFP." Chemical Society Reviews Chem. Soc. Rev. 38.10 (2009): 2821. Nobel Prize. Kungl Vetenskapsakademien The Royal Swedish Academy of Sciences, 8 Oct. 2008. Web. 2 Dec. 2015.
3. "Maltose Binding Protein Expression." New England Biolabs . N.p., n.d. Web. 2 Dec. 2015.4. "During an anion exchange chromatography experiment, 350 mM KCl in 15 mM Tris is added to elute
the protein. What is the exchange ion? Cl- , Tris-base, K+, or Tris." Socratic. N.p., 25 Feb. 2015. Web. 4 Dec. 2015.
5. Principle of coupled enzymatic assays using our Amplex® Red. Thermo Fisher Scientific, Waltham, MA. Web. 5 Dec. 2015.
6. Ni-IDA-Agarose. 2015. Gentaur BVBA, Belgium. Web. 4 Dec. 2015.