Team:ACIBADEM ISTANBUL/Improve
Table of Contents
2. Usage
3. Primers
4. Original Part
5. Our (Improved) Part
6. Characterisation
7. What experiments did we do?
Why was it Designed?
With LTNF 2.0, we finally have a viable peptide-based anti-venom that can finally replace the ancient and morbidly flawed immunoglobulin based anti-venom.
Usage
LTNF 2.0 can be used an affordable, readily available, easy to manufacture and safe treatment modality in snake endemic regions against envenomation.
Primers
We teamed up with sentegen, a leading Turkish synthetic biology company, to design and synthesize the primers required for the Biobrick part registration.
| LTNF 2.0 | Quality Control | |
| Forward Primer | (C_LTNF_C F_EcoRI) gcgcgcGAATTCATGTGTGGCGGTGGAGGC |
(BBa_G00100) tgccacctgacgtctaagaa |
| Reverse Primer | (C _LTNF_C_R_PstI) gcgcgcCTGCAGTCAACAAGGCGTACTTGG |
(BBa_G00101) attaccgcctttgagtgagc |
Original Parts
Previous studies have shown that only the first 10 amino acids are required to capture the anti-venom activity of LTNF protein.
DNA sequence
CTG AAA GCG ATG GAT CCG ACC CCG CCG CTG
Amino acid sequence
L K A M D P T P P L
Our (Improved) Part
DNA sequence
The DNA sequence was optimized for use in Pichia pastoris by using IDT’s Codon Optimization tool.
TGT GGC GGT GGA GGC TTG AAA GCA ATG GAC CCT ACC CCC CCC CTG TGG ATA AAA ACG GAG TCA CCA AGT ACG CCT TGT
Amino acid sequence
C GGGG LKAMDPTPPLWIKTESPSTP C
It contains:
Two Cysteine amino acids have been added as the first (1) and last (26) amino acids in the part. The two Cysteine amino acids form a disulphide bridge, thereby connecting the ends of the polypeptide chain together resulting in a more circularized structure, this process is called, “Circularization.”
Circularization causes an increase in entropy, resulting in an increase in stability. More stable peptides have fever toxic effects and longer half-lives compared to non-circularized ones.
Circularization can also increase the bioactivity and thus efficacy of the peptide, resulting in greater potency.
Spacer
Early modeling studies indicated that placing two Cysteine amino acids right next to the LTNF (Active site) sequence caused a great deal of strain, reducing its c-score as well as its activity.
Four Glycine amino acids were found to greatly reduce the stress found between the first cysteine amino acid and the LTNF 10 active site, whilst also avoiding interfering with its biochemical properties, given its chemically inert and simplistic nature.
LTNF 10
The original Biobrick part that displays anti-venom activity via the inhibition of metalloproteinases.
Extension
Adding a Cystiene bond to the end of the 10 amino acid LTNF sequence resulted in stress to the end part of the active site. As a way to combat this, we began adding the natural continuation of the LTNF 10 amino acids bit by bit, the highest c-score occurred when exactly an additional 10 amino acids (extensions) were added to the LTNF 10, striking a balance between the least stress and highest entropy levels.
Where did we obtain it?
LTNF is naturally found in the serum of Opossums (Didelphis virginiana). LTNF 2.0 was ordered as a peptide from Peptide 2.0 and our plasmid was designed and manufactured by Genescript, using the pPICZalphaA plasmid.
Biosafety:
Biosafety concerns were addressed in the mandatory safety form, a copy of which can be found here.
To summarise:
Pichia pastoris is a yeast strain that displays a Biosafety Level of 1.
LTNF 2.0 is even safer than the original LTNF peptide, which already displays a Biosafety level of 1.
Debugging
We encountered one slight problem with the LTNF 2.0 that was delivered by Peptide 2.0, that being a large amount of electrostatic activity causing the lyopholized peptides to stick to our weighing spatula.
This resulted in us having to wash off the particles stuck to the weighing spatula back into their containers, lyophilzing it (a process that takes 24 hours) just to be able to weigh them for our experiments.
Sean Fisher from Peptide 2.0’s Customer Service stated that the electrostatic activity is most likely due to the peptides interaction with the plastic containers they were sent with.
As a solution, they have agreed to aliquot our future peptide orders into several vials in order to avoid this problem.
Characterisation
The original LTNF has a half-life of 0.5 hours.
The half-life of LTNF 2.0 has a half-life of 14.1 hours.
LTNF 2.0 is 28.2 times more stable than the original LTNF.
Expression rate
For the predicted value, the maximum population density, specific growth rate, protein concentration, and minimum lag phase duration were 15.7 mg/ml, 3.4 h–1, 0.78 mg/ml, and 4.2 h, and the actual values were 14.3 ± 3.5 mg/ml, 3.6 ± 0.6 h–1, 0.72 ± 0.2 mg/ml, and 4.4 ± 1.6 h, respectively.
Strain selection
LTNF 2.0 was designed with industrialization in mind, and has been optimized for fermentation via the pPICZaplhaA plasmid for its synthesis in the Pichia pastoris strain of yeast.
Pichia pastoris was our yeast strain of choice for the following reasons:
2) Can be used for large scale production, making it an ideal candidate for use in the industrialization of LTNF 2.0.
3) Can be used for high-yield, high productivity protein processing.
4) Produces proteins that are not contaminated with endotoxin or viruses.
5) Pichia pastoris has already been approved for therapeutic use by the biggest regulatory agencies.
6) Pichia pastoris can be used in edibles, meaning that it can be utilized to make a consumable, prophylactic anti-venom( “Calmette Loops” idea in Human Practice) for snake handlers and people living in snake-endemic regions.
What experiments did we do?
LTNF hemotoxic activity
Controls
HPLC Peptide stability:
Our control group was the original, 10 Amino acid LTNF sequence.
LTNF hemotoxic
Our control group was the original, 10 Amino acid LTNF sequence.