Protein conjugate preparation

Protein Conjugate Preparation. Immunogen was prepared by the method of Tijssen (11) with all procedures carried out at 0°C with stirring. One-half mM of the amino substituted analog of clomazone (Figure 1) was dissolved in 15 mL 0.167 M hydrochloric acid with 0.052 M sodium nitrate added dropwise until a slight excess developed as indicated by a starch-iodide test. After 30 minutes,... 

P. De, M. Li, D.R. Gondi, and B.S. Sumerlin, Temperature-regulated activity of responsive polymer-protein conjugates prepared by grafting-from via RAFT polymerization, J Am Chem Soc, 130 (34), 11288-9, 2008. 

B. F. Erlanger, F. Borek, S.M. Beiser and S. Lieberman, Steroid-protein conjugates. Preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone, J. Biol. Chem. 228 713 (1957). 

Li, M. (2010). Stimuli-responsive polymer-protein conjugates prepared by reversible addition-fragmentation chain transfer polymerization. Dissertation. Southern methodist university, 185 pages 3418903. http //gradworks.umi.com/34/18/3418903.html. 

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 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. 

Since SIAB is water-insoluble, it must be dissolved first in organic solvent prior to addition to an aqueous reaction medium. The most commonly used solvents for this purpose include DMSO and DMF. Typically, a concentrated stock solution is prepared in one of these solvents and an aliquot added to the protein conjugation solution. Long-term storage of the reagent in these solvents is not recommended, however, due to slow uptake of water and breakdown of the NHS ester end. 

Characterizing the resultant complex for the amount of protein per liposome is somewhat more difficult than in other protein conjugation applications. The protein-liposome composition is highly dependent on the size of each liposomal particle, the amount of protein charged to the reaction, and the mole quantity of reactive lipid present in the bilayer construction. An approach to solving this problem is presented by Hutchinson et al. (1989). In analyzing at least 17 different protein-liposome preparations, the ratio of proteindipid content (pg protein/pg lipid) in most of the complexes ranged from a low of about 4 to as much as 675. In some instances, however, up to 6,000 molecules of a particular protein could be incorporated into each liposome. 

Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Ghem. 3, 2. 

King, P., Li, Y., and Kochoumian, L. (1978) Preparation of protein conjugates via intermolecular disulfide bond formation. Biochemistry 17, 1499. 

Kitagawa, T., Kawasaki, T., and Munechika, H. (1982) Enzyme immunoassay of blasticidin S with high sensitivity a new and convenient method for preparation of immunogenic (hapten-protein) conjugates. J. Biochem. (Tokyo) 92, 585-590. 

Zalipsky, S., Seltzer, R., and Nho, K. (1991) Succinimidyl carbonates of polyethylene glycol Useful reactive polymers for preparation of protein conjugates. In Polymeric Drugs and Drug Delivery Systems (R.L. Dunn, and R.M. Ottenbrite, eds.), pp. 91-100. American Chemical Society, Washington, D.C. 

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