Our project has two separate sets of experimental methods, since our original plan was to conduct research in a laboratory setting. The experimental methods are very extensive to reflect every critical step which would allow us to answer our research question. First, we would need to perform a transformation of the ErbB4 plasmid using E. coli bacteria, and then we would perform a transfection in which the ErbB4 plasmids are transferred to the mammalian cell lines. Afterwards, an SDS-PAGE and Western Blot are conducted on the cell lines. The steps for these experimental methods were provided by Professor Judith Klein-Seetharaman and Dr. Lokender Kumar from the Colorado School of Mines.
However, we were unable to conduct this experiment in its entirety, and so we shifted our project to be fully computational. The computational and experimental methods are both provided below.
Analyze various scientific papers relating to ErbB4 and TACE/ADAM17 to derive information about the proteolytic cleavage site of ErbB4
Compile the sequences of 76 known TACE substrates
a. Analyze known cleavage sites for those substrates
i. Compare cleavage site locations relative to the transmembrane domain
ii. Look for patterns in the amino acid residues around each cleavage site
iii. Look for secondary structure (using JPred) and disorder predictions (using PONDR) for known cleavages sites
iv. Predict possible TACE cleavage sites for ErbB4 (using BLAST)
Find polypeptide structures on the RCSB Protein Databank for ErbB4, and TACE.
Upload ErbB4 and TACE structures to Pymol
a. Identify potential TACE cleavage sites in ErbB4
b. Predict how the mutations will impact proteolysis of ErbB4 based on their location within its three-dimensional structure
Find DNA sequence of all three ErbB4 variants (wild-type and two mutations), add His and 1D4 tags to each sequence.
a. ErbB4 wild-type nucleotide sequence obtained from University of California Santa Cruz - (http://genome.ucsc.edu/cgi-bin/hgc?hgsid=932837491_CyYrqZiAwnWkjqvdFCaLW2gMvTuT&g=htcGeneInGenome&i=uc002veg.1&c=chr2&l=212240441&r=213403352&o=knownGene&table=knownGene)
i. link to our sequence here
b. ErbB4 wild-type amino acid sequence obtained from UniProt Database - (https://www.uniprot.org/uniprot/Q15303#sequences)
i. link to our sequence here
c. ErbB4 ALS mutation information obtained from “ERBB4 mutations that disrupt the neuregulin-ErbB4 pathway cause amyotrophic lateral sclerosis type 19,” Takahashi, 2013 - (https://pubmed.ncbi.nlm.nih.gov/24119685/)
i. link to Mutant Variant 1 (Mutation CM1311193) sequences here
ii. link to Mutant Variant 2 (Mutation CM1311194) sequences here
Transformation of ErbB4 plasmid using E. coli bacteria; amplification to produce more of the DNA using the E. coli
Transfect mammalian cells (Michigan Cancer Foundation-7 or Human Embryonic Kidney cells) with ErbB4 encoding plasmids to form groups differing by plasmid containing ErbB4 gene. Two possible procedures:
a. Using Calcium-Phosphate-based method (making the ErbB4 using the transfected DNA)
i. Reagents
HBS buffer: 2X HBS 280mM NaCl 8.18g/500ml pH with NaOH 10mM KCl 0.37g/500ml 1.5mM Na2HPO4 0.106g(anhydrous)/500ml 12mM dextrose (glucose) 1.08g/500ml 50mM HEPES pH7.05 5.96g/500ml, After adjusting volume with ddH2O, filter sterilize with 0.22µm filter
2M CaCl2: Make fresh weekly (or keep frozen) 2.94g CaCl2 / 10ml H2O Filter sterilize
Sterile water, DMEM, PBS
ii. Calcium phosphate transfection protocol
Split cells 1 🡪 5 about 24 hours before transfection (some protocols say 4-6 hours)
Take 2x HBS buffer, 2M CaCl2, DNA out of the freezer and keep at RT (takes about 2 hours to thaw)
Prepare transfection cocktail as follows:
a. Add y ul water into sterile tube
b. Add x ul 20 microgram DNA dropwise into the water and shake tube slightly (x+y=450 ul)
c. Add 50 ul of 2.5M CaCl2 dropwise into the diluted DNA and shake tube slightly between the drops
d. Close tube and shake for 10 seconds to ensure proper mixing
e. Add 500 ul of 2x HBS dropwise while shaking the tube
f. Close tube and shake vigorously for 10 sec. The mixture should get cloudy
g. Let stand at RT for one minute.
h. During this minute take the dish out of the incubator
i. Mix the cocktail via pipetting once and add dropwise into growth medium over the dish
j. Mix gently and return to incubator
Harvest the cells after 48 hours
a. Aspirate media
b. Wash with 4 ml PBS
c. Add 2 ml PBS
d. Scrape off the cells with scraper and transfer to Falcon tube
e. Pellet cells for 10 min at 3000 rpm and 4oC
b. Using DEAE-Dextran Method
i. Reagents
a. 10x DEAE-dextran 2.5mg/ml sterile
b. 10x Tris-Hcl 1 M pH 8 sterile
c. DMEM
d. DMEM+++
e. All solutions at 37C
12.5 microgram plasmid-DNA per 15 cm dish or 10 microgram per 10 cm dish is added to 10 ml (15 cm) or 6 ml (10 cm) of 0.25mg/ml DEAE-dextran, 0.1M Tris-HCl pH 8 in DMEM
Remove media from plates
Wash with DMEM twice (approx. 5 ml for 10 cm, or 10 ml for 15 cm)
Add mixture from 1 and place plate into incubator for 6 hours
Remove DNA
Add 15 ml of 0.1mM chloroquin in DMEM to 15 cm plate or 6 ml to 10 cm plate
Incubate for 2 hours
Remove chloroquine
Wash twice with DMEM
Add 20 ml DMEM+++ per 15 cm dish or 10 ml DMEM+++ per 10 cm dish
ii. Freeze-down of cells for long term storage
Trypsinize a plate
Spin at 1000 rpm for 10 min
Discard the supernatant
Resuspend pellet in 5 ml of 10% DMSO in DMEM+++
Place 1 ml aliquots in cryogenic storage vials (Corning or Sarstedt)
Place vials in Styrofoam boxes (Sarstedt or other) or dedicated box with isopropanol coating
Place box in -70oC freezer overnight (cooling rate 1oC/min)
Transfer the cells to liquid nitrogen storage anytime after that
Incubate cells from 24-72 hours (varying times).
Test time course of ErbB4 expression between wild-type sequence and mutant sequences using Western Blot
a. Each gel separated based on stain being used (i.e., one for Anti-His tag stain, one for 1D4 stain, one for anti-ErbB4 stain)
b. Transfer samples from each group into wells in a gel. Each gel will include a molecular marker lane, ErbB4 wildtype, and each ErbB4 mutant variant.
c. Place gels in container with buffer solution and begin electrophoresis (protein fragments will be separated by mass as they slowly travel through the gel)
d. Remove gels from container
e. Stain antibodies for His and 1D4 tags and add 1 type to each gel. Fragments containing their epitopes will now be easily identified and comparable between the samples in each well
Take images of each gel for comparison
Graph trends in results and record data in a table
If no significant differences in proteolysis between the wild-type and mutant sequences are identified, will then add ligand NRG1 (to induce cleavage) in varying concentrations and test its impact on proteolysis (repeat steps 5-7)
a. Will use four concentration amounts: no ligand, small concentration, medium concentration, large concentration
b. Will test each differing concentration on ErbB4 wild-type protein (control), ErbB4 mutant variant 1, and ErbB4 mutant variant 2
If no significant differences have been observed yet, experiment will be repeated with a controlled amount of TACE/ADAM17 added to each group to induce cleavage of ErbB4 proteins (repeat steps 5-7)
All of the COVID-19 precautions implemented by the Colorado School of Mines will be observed over the course of the experiment. These precautions include wearing masks at all times, sterilizing work surfaces often, and maintaining the recommended 6-foot distance whenever possible.
If at any point the Colorado School of Mines deems it unsafe for our research to be conducted on campus, we will move to computational analysis of ErbB4, which will be carried out from our homes using online software.
Personal Protective Equipment:
We will be wearing face masks, safety goggles, and disposable nitrile gloves at all times. All areas and surfaces will be frequently disinfected with 70% ethanol. All cell cultures will be handled in a biosafety cabinet.
Other Safety Precautions:
The safety precautions associated with potentially hazardous biological agents and hazardous chemicals/activities/devices are detailed below.
Human ErbB4 Gene (in mammalian expression vector)
Human ErbB4 gene for wild-type and two point mutations including HIS and 1D4 tags. Vector does not contain resistance genes and will only be used for transient transfection. Low hazard.
Human Embryonic Kidney Cells (HEK-293)
Michigan Cancer Foundation Cells (MCF-7)
The human ErbB4 gene in the mammalian expression vector will be used to transfect cells (HEK-293 and MCF-7). Because the HEK-293 and MCF-7 are mammalian cell cultures, they are classified as Biosafety Level 2 (BSL2). Colorado School of Mines and the lab that will be used are BSL2 approved. The laboratory has sink availability and separate entrances. We will have BSL2 training prior to experimentation, and all work with cells will be conducted inside a biological safety cabinet under sterile conditions. All waste materials will be disposed of in accordance to BSL2 biohazard sterilization conditions.
E. coli
E. coli will be used for amplification of the ErbB4 plasmid. Very low risk: BSL1.
NRG1
NRG1, ligand for ErbB4, will be a commercially obtained purified protein. Ligand will be added to HEK-293 or MCF-7 cells. Very low risk: BSL1.
TACE
TACE, protease for ErbB4, will also be a commercially obtained purified protein. Protease will be added to HEK-293 or MCF-7 cells. Very low risk: BSL1.
*add bleach to solutions; all bagged in biohazard bags for sterilization
Overall, there will be a low risk associated with this experiment. All chemicals used are bio-compatible buffer systems, and there are no hazardous chemicals being used other than the biohazards listed above. For instrumentation, we will be using a gel apparatus (SDS-PAGE), Western Blot transfer and imaging equipment, and a CO₂ incubator for encouraging cell growth. None of these devices are considered hazardous.
Additionally, we will be supervised by a qualified scientist at all times to ensure that all chemicals and devices are used properly and safely.