Aug 11, 2020
Conjugation of peptide fragment 579-601 of HIV-gp41 or fragments 308-331 or 421-438 of the HIV-gp120 with keyhole limpet hemocyanin (KLH).
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Conjugation of peptide fragment 579-601 of HIV-gp41 or fragments 308-331 or 421-438 of the HIV-gp120 with keyhole limpet hemocyanin (KLH).. protocols.io https://dx.doi.org/10.17504/protocols.io.bjhukj6w
Manuscript citation:
Angel Justiz-Vaillant. Conjugation of peptide fragment 579-601 of HIV-gp41 or fragments 308-331 or 421-438 of the HIV-gp120 with keyhole limpet hemocyanin (KLH)..protocols.iohttps://dx.doi.org/10.17504/protocols.io.bjhukj6w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 10, 2020
Last Modified: August 11, 2020
Protocol Integer ID: 40212
Abstract
Chemical synthesis facilitates the generation of peptides which are very difficult to express in bacteria, peptide/protein backbone modification, the incorporation of unnatural amino acids, and the production or synthesis of D-proteins.
The C-terminal cysteine can be added to the amino acidic sequences of HIV peptides (fragment 579-601 of the HIV-gp41 and fragments 308-331 or 421-438 of the HIV-gp120). These peptide fragments were dimerized by cysteine oxidation with dimethyl-sulfoxide [1] to faciliutate their conjugation to keyhole limpet hemocyanin that acts as a carrier protein.
Reference:
1. Tam JP, Wu CR, Liu w, Zhang JW (1991) Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications. J Am Chem Soc 113: 66576662.
Guidelines
The Protocol has a high level of reproducibility and has worked for many other HIV peptides.
Materials
MATERIALS
Glutaraldehyde EM Grade 25%Sigma AldrichCatalog #G5882-50ML
10mg KLH (Keyhole Limpet Hemocyanin) (Immunological Grade)G-BiosciencesCatalog #786-088
Peptide 579-601 of HIV-gp41
Peptide 308-331 HIV-gp120
Peptide 421-438 HIV-gp120
Safety warnings
Side effects include skin irritation, nausea, headache, and shortness of breath.
Before start
Ensure that the HIV peptide requires a terminal cysteine, so a disulfide bond formation in the peptides by dimethyl sulfoxide is needed before the conjugation to KLH. For example, fragment 254-274 of the second conserved domain of HIV gp120 has a terminal cysteine, and this peptide can be conjugated to KLH without previous treatment with dimethyl sulfoxide.
These peptide fragment (579-601 from HIV-gp41) is dimerized by cysteine oxidation with dimethyl-sulfoxide. The HIV peptide is dissolved in 5% acetic acid to a final concentration of 5.1 mg/ml.
The pH of the medium is adjusted to 6 with 1 M (NH4)2CO3.
Dimethyl-sulfoxide is added to 20% of the final volume, and after four hours at room temperature (RT), the solute is extracted.
Then, the peptide is dissolved in 3 ml 5% trifluoroacetic acid and precipitated with 35 ml cold ether.
The precipitate is dialyzed against 1.2 liters of deionized water, pH 7 at 4°C overnight.
Then, 1 mg of keyhole limpet hemocyanin (KLH) is diluted in 2.1 ml 0.1 M borate buffer (1.24 g boric acid, 1.90 g sodium tetraborate, pH 10, in 500 mL deionized water).
In a 20 ml glass tube, with a gentle stirring, 1.1 µmol of the HIV synthetic peptide (with C-terminal cysteine added) and 0.22 mL 0.3% glutaraldehyde solution (ACS reagent grade, pH 5.5, Sigma-Aldrich) are slowly mixed at RT and left to stand for 1.50 hrs.
When a yellow coloration is observed this indicates that the conjugation process is successful.
To blocking the excess of glutaraldehyde, 0.26 ml of 1 M glycine (Sigma-Aldrich) is added.
The mix is left for 32 min at RT.
The HIV-hemocynin conjugate is then dialyzed against 1.3 liters 0.1 M of borate buffer, pH 8.4 through the night at 4°C.
Then the previous buffer is used to dialyze the preparations for 8 hrs at 4°C.
The dialysates is stored at 4°C until further use.