oxytocin-neurophysin i results nec i cleavage enzyme

Adam Christiansen, Amrinder Grewal, Austin Kothig, Brianna Carrels, Brooke Heatherington, Carissa Kirk, Cassandra Logue, Chris Isaac, Dylan Sutherland, Elaine Bird, Erin Kelly, Fiona Spitzig, Hans-Joachim Wieden,

Isaac Ward, Joseph Adams, Katie Thomas, Kieran McCormack, Krista Fjordbotten, Mackenzie Coatham, Marie Cooney, Mark Hasell, Patrick O’Donnell, Tessa Carrels, Thomas Kazakoff, Wesley Mosimann, Yoyo Yao

Introduction

• Continue development of our Oxytocin-Neurophysin I and NEC I systems • Further characterize our Oxytocin-Neurophysin I construct • Complete the assembly and characterization our NECI construct • Continue to optimize the NECI construct investigating the efficiency of promoters and ribosomal binding sites • Further our involvement in the community, particularly with school presentations • Give follow up presentation to Lethbridge School District No. 51 regarding our progress • Continue producing informative videos of lab practices for high school students

Math Model Our mathematical model was designed to indicate the amount of protein produced by any number of E. coli cells as a function of time. Accordingly, bacterial growth is included to facilitate calculation of the total protein produced by the cells. The next expression represents the total mRNA produced by each cell assuming that it is used for translation nearly instantly. Next, our expression is multiplied by the Translational Constant derived from the speed of transcription versus the speed of translation.

A more comprehensive explanation can be found on the Lethbridge High School 2013 Wiki.

Speed of Transcription

mRNA Decay

Bacterial Growth

Speed of Translation

Translational Constant

Gene Length / Protein Length

Future Directions

OXYTOCIN-NEUROPHYSIN I Naturally, Oxytocin has a signal sequence in mammalian cells. For our construct we are using the PelB signal sequence as a substitute for the original signal sequence. The signal sequence is cleaved after it moves through the inner membrane and into the periplasmic space. When it is cleaved off, it allows for the first cysteine to create a disulfide bond with another cysteine facilitating the proper folding of Oxytocin. We also used nickel-sepharose to purify our histidine tagged construct that is present in prokaryotic cell cultures since it can easily be extracted from solution by the string of histidine residues that bind to the nickel ions.

RESULTS

NEC I CLEAVAGE ENZYME The cleavage enzyme, Neuroendocrine Convertase 1 (NEC1), separates Neurophysin from Oxytocin allowing it to bind to its receptors. In order to determine the optimal promoter to produce NEC1 at a rate complementary to the production of prepro-oxyphysin, we attempted to use mCherry as a substitute for NEC1 during fluorescence assays. Since NEC I, the cleavage enzyme, is so big in size, to save time and resources we used the construct mCherry to simulate the amount of enzyme needed to cut our Oxytocin-Neurophysin I construct. Tests would also provide further information about the construct mCherry with a promoter, as currently, it is not a part in the parts registry.

Fig. 2. Oxytocin-Neurophysin I construct showing modified Oxytocin Gene

Fig. 3. Neuroendocrine Convertase I construct with added histidine tags

Fig. 1. Oxytocin’s polypeptide chain. The disulfide bond between C1 and C6 is shown.

Fig.5. 1% Gel electrophoresis showing K314100_Oxytocin-Neurophysin in lanes 5, 8, 10, and 12. The second band in lane 3 shows our promoter (518bp). The second band in lane 5 shows isolated Oxytocin (in black circle, 406bp). The band in lane 6 is the plasmid pSB1C3 (2070bp). In lane 7 you will find our full construct (2974bp). Lanes 8, 10, and 12 shows K314100_Oxytocin-Neurophysin I cut with EcoRI and PstI.

Fig. 6. Slot Blot test showing histidine tag inclusion The Slot Blot confirms the presence of a Oxytocin-Neurophysin I. The primary antibody is sensitive to histidine and will bind to it. The secondary antibody is sensitive to the primary antibody, with the chemiluminescent enzyme horseradish peroxidase attached to it. Should the secondary antibody bind to the primary antibody, they will both remain attached to the histidine tag on the protein and when induced with luminol, p-coumaric acid and hydrogen peroxide, will glow.

Oxytocin is a neurohypophyseal hormone which, along with its carrier molecule Neurophysin I, is produced in mammals. Oxytocin has a half life of 3-10 minutes and Neurophysin I is used to stabilize it, increasing its shelf life1. Together, they form the complex prepro-oxyphysin. Oxytocin is cleaved from Neurophysin I by the enzyme Neuroendocrine Convertase I (NEC I). Oxytocin aids in contractions during birth, as well as pair bonding, maternal behaviour, and the secretion of milk from a mother during breastfeeding2. Recent studies have shown that irregular levels of Oxytocin are a potential cause of several social disorders, including autism, schizophrenia, and overly aggressive behaviour3,4. Some scientists claim that Oxytocin could be used to treat these disorders5. However, many properties of Oxytocin are still unknown and use for treatment is controversial5. Our goal was to produce a stable form of natural Oxytocin, by using the bacteria Escherichia coli to produce prepro-oxyphysin and NEC I in separate cell systems. We can then use NEC I to release Oxytocin from Neurophysin I when we need it. We hope to make it more available to researchers to study and look into potential treatment uses.

Oxytocin-Neurophysin I and NEC I Cleavage Enzyme

Lane Contents Vol.

1 Protein Molecular Weight Marker 20μL

2 Cbf5-Nop10-Gar1 20μL

3 Buffer A wash 30μL

4 Buffer B wash 30μL

5 Buffer E wash 30μL

6 S-30 S/N 30μL

7 Buffer E – concentrated prepro-oxyphysin

30μL

Lane Contents Vol.

1 1KB DNA LADDER 3μL

2 K314100_pSB1C3 cut EcoRI 15μL

3 K314100_pSB1C3 cut EcoRI and PstI 15μL

4 Oxytocin-Neurophysin I_PUC57a cut EcoRI 15μL

5 Oxytocin-Neurophysin I_PUC57a cut EcoRI and PstI

15μL

6 Gel Extraction of pSB1C3 cut EcoRI and PstI 15μL

7 K314100_Oxytocin_Neurophysin I cut EcoRI 15μL

8 K314100_Oxytocin_Neurophysin I cut EcoRI and PstI

15μL



15μL



15μL

Lane Contents

1 100μL Oxytocin in 200μL TBS



4 300μL Oxytocin

5 25pM TruB-His positive control

1. Rydén, G & Sjöholm, I. (1969) Acta Endocrinol. Half-life of Oxytocin in blood of pregnant and non-pregnant women. Vol. 61: 425-431 2. Shahrestani, S. et al. (2013) The Impact of a Single Administration of Intranasal Oxytocin on the Recognition of Basic Emotions in Humans: A Meta-Analysis. http://www.ncbi.nlm.nih.gov/pubmed/23575742 (accessed on June 22, 2013) 3. Tenq, B. L. et al. (2013) Neuropharmacology. Prosocial effects of oxytocin in two mouse models of autism spectrum disorders. Vol. 72: 187-196 4. Heinrichs, M & Domes, G.(2008) Progress in Brain Research. Neuropeptides and social behaviour: effects of oxytocin and vasopressin in humans. Vol. 170: 337-350 5. Miller, G. (2013) Science. The Promise and Perils of Oxytocin. Vol. 339: 267-269

References

5 12 10 8 3 6 7

Fig. 4. Silver Stain 16.5% Tris-Tricine PAGE with orange outlines to indicate suspected prepro-oxyphysin bands. Red outlines Nop10 (7.2 kDa). Lowest visible band in Protein Molecular Weight Marker is 14 kDa. prepro-oxyphysin is approximately 10kDa, between 14kDa and 7.2kDa, which is where our suspected prepro-oxyphysin bands are.

oxytocin-neurophysin i results nec i cleavage enzyme

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