The combined molecular weights show where the band for NS5A and Elongin C would occur due to the TEV linker. Our laboratory worked with the viral protein, NS5A, to study its interactions with host proteins, Elongin B and Elongin C, which are part of the Cullin-RING ligase (CRL) complex of the. Hepatitis C virus (HCV), first identified in 1989, is a positive-strand single-stranded RNA virus of the family Flaviviridae and genus Hepacivirus.1 Such viruses have a genome consisting of ribonucleic acid that acts as messenger -RNA can act and be directly translated into proteins using the host cells' ribosomes.
Symptoms include fever, fatigue, abdominal pain, loss of appetite, nausea, vomiting, joint pain, and jaundice.2 Some people may experience no symptoms at all and are diagnosed incidentally through elevated alanine aminotransferase levels in routine blood work, which indicates hepatic injury. and tissue necrosis.1 HCV has seven genotypes with 67 different subspecies; genotype 1a and 1b are most common in the United States.1,2 Genotype 3 is most common in India, genotype 4 is most common in Africa and the Middle East, and genotype 6 is most common in Southeast Asia and Africa. South. 1 HCV genotypes are area-specific, making it much more difficult to treat or eradicate globally. Viral production of HCV is very high, producing 1010-1012 virions per cell per day.1 The genome of the virus also changes frequently, due to the speed of the virus. Acute Hep C can be treated with antivirals, depending on the genotype of the virus contributing to the infection.
However, chronic Hep C infection may require liver transplantation.2 Because of the high prevalence of liver transplants and cancer, it is important to find more effective therapies. This process occurs in a three-step procedure starting with a ubiquitin-activating enzyme (E1), then a ubiquitin-conjugating enzyme (E2), and finally a ubiquitin protein ligase (E3).5 Most common. Through sequence comparison, it was found that NS5A, a protein found in the Hepatitis C virus, has a sequence similar to that of the SOCS box binding domain (Figure 4).
The RMSD is the measure of the average distances between the atoms of the two superimposed proteins; the lower the RMSD, the better the superposition of the two molecules.
Cell Biology Techniques
Liquid Media Creation
Bacterial Plating
Overnight Cultures
Glycerol stock creation
Plasmid Construction
- Test digests of plasmids
- PCR, Digest, and Purification of Inserts
- Ligation
- Transformation into bacterial cell lines
- Agarose Gel Electrophoresis
PCR products were also digested, but in this process the digests were left in the thermocycler for two hours instead of 30 minutes. After that, the products were removed from the thermocycler and treated with alkaline phosphatase, Fast AP from ThermoFisher, to cleave the phosphate ends of the inserts. They were again placed in the thermocycler at 37°C for 10 min and purified using the Promega Wizard SV Gel and PCR purification system.
The amounts of primer and vector required to obtain a 3:1 ratio of primer to vector were calculated using the NEBio Ligation Calculator for ligation. The cell solution was mixed gently and 50 µL of cells were pipetted into a 15 mL culture tube. A concentration of 1 pg to 100 ng of plasmid DNA was added to the cell mixture and the tube was shaken to mix the cells and DNA.
The mixture was placed on ice for 30 minutes and then heat shocked at exactly 42°C for 30 seconds. Then, 950 µL of LB medium was pipetted into the mixture, which was placed at 37°C for one hour on the shaker. The cells were mixed, then 200 µl were plated on LB media with 50 µg/ml ampicillin and cultured overnight.
This was done by thawing 50 µL of ThermoFisher One Shot cells and pipetting 2 µL of each plasmid into separate cell tubes. Then, after the mixture cooled a bit, 1x Syber Safe DNA gel stain was added, and the gel was poured into the gel mold.
Protein Interaction Analysis
Protein Expression
Immobilized Metal Affinity Chromatography
10 ml of lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 5 mM B-mercaptoethanol and protease inhibitor tablet) was added per gram of bacterial cell pellet. Lysozyme was added at a concentration of 2 mg/ml and the mixture was incubated for 20 minutes. The mixture was sonicated to lyse the cells at 30% amplitude for five 10 second intervals with 50 seconds between each pulse.
Cells were then spun down at 6000 rpm for 20 min and filtered using a 0.45 µL filter and syringe. This filtrate was added to a Ni-NTA spin column (ThermoFisher) in three 5 mL increments, each incubated for 10 min before spinning for two min at 700 x g. The supernatant flow-through was collected each time and then the column was washed three times with 2 mL of lysis buffer, spun for two minutes at 700 x g.
SDS-PAGE Analysis
The gel was removed from the apparatus and plastic wrap and placed in Coomassie stain. The gel was then placed in destain overnight, or until there was sufficient contrast between the bands and the gel.
Plasmid Construction
This table was created to help simplify the different strains and plasmids eluted in the results section of this thesis. Chimeras were generated through molecular cloning techniques, such as digestion, insert purification, and ligation. Several consecutive digestions were done to end up with DN and DS plasmids.
Plasmid B was the first digested plasmid to insert the genes for NS5A or SOCS2 into the multiple cloning site 2 (MCS2). Both restriction enzymes used to insert these genes (Table 3) include a 6-His tag for labeling proteins of interest. Plasmids CS and CN were further digested to insert the gene for Elongin C into MCS2.
Protein Expression
After construction of plasmids DN and DS, these plasmids were transformed into -dam/-dcm cells for expression experiments.
Immobilized Metal Affinity Chromatography (IMAC)
The combined molecular weights indicate where the band for SOCS2 and Elongin C would appear due to the TEV linker. This was done to test the effects of removing the TEV linker on the appearance of proteins in SDS-PAGE analysis. However, even with the TEV linker cut off, no bands appeared on the SDS-PAGE gel.
The data in Figures 7 and 9 show that the proteins were not toxic to the cells during expression. There was no problem with protein expression, and this could be ruled out as something that may have gone wrong in the study. It was hypothesized that NS5A, due to its similar sequence to the SOCS box-binding domain, would bind to host proteins Elongin B and Elongin C (as SOCS2 does), disrupting the endogenous ubiquitination process.
Through immobilized metal affinity chromatography (IMAC), the 6-His tag on NS5A should bind to the nickel resin of Ni-NTA columns, causing Elongin B and Elongin C to also remain on the resin by binding to NS5A. This would cause all three proteins to appear in the elution fractions when analyzed by SDS-PAGE. 24. bands appeared in elution samples that were loaded on SDS-PAGE in both control and experimental.
Proteins were found in the insoluble pellet and the flow-through, characteristic of contaminating proteins without the 6-His tag. SOCS2 is known to bind to Elongin B and Elongin C, so there should have been bands at least showing up on this gel.8 The fact that there was not gives rise to three possible conclusions: 1) this is not a successful method in showing .. interaction between these proteins, 2) our proteins are not soluble in our elution buffer, or 3) the proteins were too diluted to show up on the gel. There is also a possibility that the TEV linker may restrict the protein from binding to the resin or interacting with the target proteins.
The TEV linker can bind the 6-His tag, preventing it from binding to the resin on the column, causing our target proteins to wash through the column during washes or flow-through. If our proteins are not soluble in the elution buffer, it will not pull them down into the elution fractions, meaning that the proteins may still be stuck to the resin. This can be further assessed by boiling the resin with loading buffer for SDS-PAGE analysis.
Additional studies should be conducted to correct any issues that may have arisen in the studies above. This is an important first step in the research that can be done to further test the hypothesis that NS5A binds to Elongin B and C to hijack host cell ubiquitination.
