Peptide-protein conjugates

Gauthier MA, Klok HA (2008) Peptide/protein-polymer conjugates synthetic strategies and design concepts. Chem Commun 23 2591-2611... 

Some reports even indicate that the conjugation of proteins or peptides with carbohydrates can increase dramatically their activity compared to that of the native state (Susaki et al., 1998). Carbohydrates also can provide a protective effect on modified peptides toward proteolytic digestion (Rudd et al., 1994) or mask recognition of a peptide by the immune system (Harding et al., 1993). The creation of neoglycoproteins thus can affect the activity of peptides and proteins, which are not normally glycosylated in vivo. 

Dissolve the molecule to be coupled in the same buffer used in step 1. For small molecules, add them to the reaction in at least a 10-fold molar excess to the amount of protein present. If possible, the molecule may be added directly to the protein solution in the appropriate excess. Alternatively, dissolve the molecule in the buffer at a higher concentration, and then add an aliquot of this stock solution to the protein solution. In the example of preparing a peptide-protein conjugate, dissolve the peptide in 0.1 M MES, pH 4.7, at a concentration of up to 2 mg/500 pi. 

Add the solution prepared in step 2 to the protein solution to obtain at least a 10-fold molar excess of small molecule to protein. In the case of the peptide-protein immunogen conjugate, add the 500 pi of peptide solution to the 200 pi of protein solution. 

Add EDC (Thermo Fisher) to the above solution to obtain at least a 10-fold molar excess of EDC to the protein. Alternatively, a 0.5-0.1 M EDC concentration in the reaction mixture usually works well. To make it easier to add the correct quantity of EDC, a higher concentration stock solution may be prepared if it is dissolved and used immediately. To prepare the peptide-protein conjugate, add the solution from step 3 to 10 mg of EDC in a test tube. Mix to dissolve. If this ratio of EDC to peptide or protein results in precipitation, scale back the amount of carbodiimide addition until a soluble conjugate is obtained. For some proteins, as little as 0.1 times this amount of EDC may have to be used to maintain solubility. 

This chapter describes the design, preparation, and use of hapten-carrier conjugates used to elicit an immune response toward a coupled hapten. The chemical reactions discussed for these conjugations are useful for coupling peptides, proteins, carbohydrates, oligonucleotides, and other small organic molecules to various carrier macromolecules. The resultant conjugates are important in antibody production, immune response research, and in the creation of vaccines. 

Atassi, M.Z., and Manshouri, T. (1991) Synthesis of tolerogenic monomethoxypolyethylene glycol and polyvinyl alcohol conjugates of peptides.. Protein Chem. 10, 623-627. 

Iwai, K., Fukuoka, S.-I., Fushiki, T., Kido, K., Sengoku, Y., and Semba, T. (1988) Preparation of a verifiable peptide-protein immunogen direction-controlled conjugation of a synthetic fragment of the monitor peptide with myoglobin and application for sequence analysis. Anal. Biochem. 171, 277-282. 

For the synthesis of the peptide-protein conjugates, peptides with a readive linker group, ie a maleimido group at the N-terminal amino function, were required. The synthesis of these peptides... 

Besides being presently the most efficient method for site-directed interchain disulfide bridging of two different cysteine peptides, this procedure is also recommended for a controlled peptide-protein conjugation by reacting Npys-protected cysteine peptides with properly thiol-functionalized protein carriers. 1761 In this conjugation procedure the amount of peptide grafted to the protein is quantitatively determined by measuring spectro-photometrically at 430 nm the amount of 3-nitropyridine-2(l//)-thione liberated in the reaction. An example of this approach is outlined in Scheme 18. 

Antibody, enzyme, peptide, protein, or thiolated oligonucleotide to be conjugated to avidin. 

In this unit, Basic Protocol 1 presents a procedure using casein as substrate. The Alternate Protocol describes the modification of this procedure for use with a denatured hemoglobin substrate. Basic Protocol 2 presents a procedure using a chromaphore-conjugated casein derivative, azocasein. For quantitation, the authors have chosen to use either the BCA-based colorimetric assay unitbli) for soluble protein/peptides (in Basic Protocol 1) or the intrinsic absorbance of the chromaphore-conjugated peptide products (in Basic Protocol 2). 

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