Putrescine oxidase (PuOx) is an enzyme that catalyzes the oxidation of putrescine. PuOx was purified using two different tags, GFP and MBP. GFP-PuOx purification using a Q-sepharose column was unsuccessful. MBP-PuOx purification using nickel affinity chromatography was more effective. Kinetic analysis found the Kcat and Kcat/Km of purified MBP-PuOx. FAD cofactor content analysis found a 1:1.68 ratio of FAD to PuOx monomer.
Proteins play crucial roles in nearly all biological processes. These many functions of proteins are a result of the folding of proteins into many distinct 3D structures.
Protein analysis tries to explore how amino acid sequences specify the structure of proteins and how these proteins bind to substrates and other molecules to perform their functions.
Protein analysis allows us to understand the function of the protein based on its structure.
The electrosomes, a novel surface-display system based on the specific
interaction between the cellulosomal scaffoldin protein and a cascade of
redox enzymes that allows multiple electron-release by fuel oxidation. The
electrosomes is composed of two compartment:(i) a hybrid anode, which
consists of dockerin-containing enzymes attached specifically to cohesin sites
in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid
cathode, which consists of a dockerin-containing oxygen-reducing enzyme
attached in multiple copies to the cohesin-bearing scaffoldin.
Substrate level phosphorylation and it's mechanism || Biochemistry || B Pharmacy || Project || slideshare || biology || chemistry
*images use in this ppt is only for educational purpose
In this presentation, i tell about substrate level phosphorylation
Phosphorylation involves the transfer of phosphate
group from one compound to other.
➢ Substrate level phosphorylation is a direct
phosphorylation of ADP with a phosphatase group by
using the energy obtain from a coupled reaction.
➢ Occurs in cytoplasm ( glycolysis – due to aerobic and
anaerobic condition) and in mitochondrial matrix ( krebs
cycle – anaerobic condition)
Proteins play crucial roles in nearly all biological processes. These many functions of proteins are a result of the folding of proteins into many distinct 3D structures.
Protein analysis tries to explore how amino acid sequences specify the structure of proteins and how these proteins bind to substrates and other molecules to perform their functions.
Protein analysis allows us to understand the function of the protein based on its structure.
The electrosomes, a novel surface-display system based on the specific
interaction between the cellulosomal scaffoldin protein and a cascade of
redox enzymes that allows multiple electron-release by fuel oxidation. The
electrosomes is composed of two compartment:(i) a hybrid anode, which
consists of dockerin-containing enzymes attached specifically to cohesin sites
in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid
cathode, which consists of a dockerin-containing oxygen-reducing enzyme
attached in multiple copies to the cohesin-bearing scaffoldin.
Substrate level phosphorylation and it's mechanism || Biochemistry || B Pharmacy || Project || slideshare || biology || chemistry
*images use in this ppt is only for educational purpose
In this presentation, i tell about substrate level phosphorylation
Phosphorylation involves the transfer of phosphate
group from one compound to other.
➢ Substrate level phosphorylation is a direct
phosphorylation of ADP with a phosphatase group by
using the energy obtain from a coupled reaction.
➢ Occurs in cytoplasm ( glycolysis – due to aerobic and
anaerobic condition) and in mitochondrial matrix ( krebs
cycle – anaerobic condition)
Presentation on increased bioavailability of breviscapine via a pluronic p85 ...2503jyoti
Breviscapine is a hydrophobic drug used in cerebovascular diseases.It's bioavailability due to it's efflux by P-gp system.pluronics are non-ionic tri-bolic co-polymers made up of central part of PPO(hydrophobic) which is flanked by two PEO(hydrophilic) molecules
A simple outline of oxidative phosphorylation.. It explains the process, site of occurrence, components involved, source of electron carriers and inhibitors of the process.
Presentation on increased bioavailability of breviscapine via a pluronic p85 ...2503jyoti
Breviscapine is a hydrophobic drug used in cerebovascular diseases.It's bioavailability due to it's efflux by P-gp system.pluronics are non-ionic tri-bolic co-polymers made up of central part of PPO(hydrophobic) which is flanked by two PEO(hydrophilic) molecules
A simple outline of oxidative phosphorylation.. It explains the process, site of occurrence, components involved, source of electron carriers and inhibitors of the process.
Ureases (EC 3.5.1.5), functionally, belong to the superfamily of amidohydrolases and phosphodiesterase.
Nickel containing metalloenzyme.
Ureases are found in numerous bacteria, fungi, algae, plants, and some invertebrates, as well as in soils, as a soil enzyme.
Not synthesized by animals.
James B. Sumner in 1926, Noble Prize in Chemistry in 1946.
Urease catalyzes the hydrolysis of urea to from ammonia and
Carbon dioxide
2. 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
3. Structure and Mechanism
• Putrescine Oxidase exists as is a homodimer
• Molecular Weight as homodimer: 198 kDa
• putrescine + O2 + H2O = 4-aminobutanal + NH3 + H2O2
4. 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
6. 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.
7. • 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
8. 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
9. 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 𝜇L of each peak were recovered
10. 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
11. 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
12. 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 kDa
17 kDa
28 kDa
38 kDa
49 kDa
62 kDa
98 kDa
188 kDa
6 kDa
Fractions
13 2
4 kDa
13. 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
14. 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
16. 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
17. 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 stage16 kDa
17 kDa
28 kDa
38 kDa
49 kDa
62 kDa
98 kDa
188 kDa
6 kDa
18. Conclusion
• Purified MBP-PuOx
• Determined Kinetics of purified MBP-PuOx
• Determined FAD cofactor Content of MBP-PuOx
• Learned and improved different laboratory protocols
19. Bibliography
1. 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.
Editor's Notes
Over-expression addition is measured by doing a A600
Oxidation of glucose by glucose oxidase results in generation of H2O2, which is coupled to conversion of the Amplex® Red reagent to fluorescent resorufin by HRP.
INTRODUCE USE OF MBP-PUOX before moving onto next slide
Confirmed with Kansas that they were having issues with transforming and growing the GFP-PuOx
Kcat/Km off by so much due to Km being much higher than the literature value of 3.5 [uM]
high concentration is what caused such a large reading in the elutant sample
