Subject(s)
Drug Design , Immunoglobulins/chemical synthesis , Protein Engineering , Animals , Humans , Protein Engineering/methodsABSTRACT
Three methods of synthesis of immunoglobulin conjugates with malate, lactate and glucose-6-phosphate dehydrogenases, involving the sodium metaperiodate oxidation of immunoglobulin carbohydrate component, use of water-soluble carbodiimide and the one-step glutaraldehyde technique, were compared. The glutaraldehyde method was shown to give immunoglobulin-dehydrogenase conjugates with high catalytic and immunochemical activity, which may be useful for enzyme-immunoassay.
Subject(s)
Immunoenzyme Techniques , Immunoglobulins/chemical synthesis , Oxidoreductases/chemical synthesis , Chemical Phenomena , ChemistryABSTRACT
A protected tridecapeptide, representing a new peptide corresponding to residues 56-68 of the VH domain in the mouse M603 myeloma protein, has been prepared by solid phase peptide synthesis. The protected tridecapeptide was prepared using the photolabile 4-bromomethyl-(3-nitro)-benzamidomethyl-resin and the multidetachable 2-[4-bromomethyl)phenylacetoxy]propionyl-resin as solid supports. The synthetic protocol and protecting groups were the same for both syntheses. The protected tridecapeptide was removed photolytically from both supports and the sequence integrity was determined by preview analysis using the solid phase Edman degradation procedure. The protected tridecapeptide-OMPA was purified to homogeneity by DMF/H2O precipitation and LH-60 chromatography. The purity of the protected peptide was further demonstrated by high pressure liquid chromatography on the free peptide after HF deprotection. The protected tridecapeptide was reattached to 4-bromomethyl-(3-nitro)-benzamidomethyl-resin to give the photolabile Boc-(protected)peptidyl-4-oxymethyl-(3-nitro)benzamidomethyl-resin in 25% yield. The protected tridecapeptide-oxymethylphenylacetic acid derivative was reattached to aminomethyl-resin to give Boc-(protected)peptidyl-2-[4-oxymethyl)phenyl]acetamidomethyl-resin in 45% yield and to 2-bromopropionyl-resin generating the multidetachable Boc-(protected)peptidyl-2-[(4-oxymethyl)phenylacetoxy] propionyl-resin in 80% yield. The reactivity of these reattached peptides was demonstrated by the quantitative coupling of Boc-leucine to the protected peptide-resin. The advantages and disadvantages of the different resins with respect to solid phase fragment synthesis are discussed.
Subject(s)
Immunoglobulins/chemical synthesis , Myeloma Proteins/chemical synthesis , Peptide Fragments/chemical synthesis , Amino Acid Sequence , Amino Acids/analysis , Animals , Chemical Phenomena , Chemistry , Chromatography/methods , Immunoglobulins/isolation & purification , Mice , Myeloma Proteins/isolation & purification , Peptide Fragments/isolation & purification , Photolysis , Resins, SyntheticABSTRACT
Semisynthesis provides a flexible approach for using chemical synthesis to produce large, biologically active polypeptides and analogues. Currently developing improvements in the basic methods used, including polypeptide fragmentation, peptide synthesis, and reconstitution of synthetic and native components, make this overall approach applicable to a variety of species. Sequence modification through semisynthesis thus provides a flexible route to explore the code of rules whereby primary structure directs higher order properties of folded conformation and biological function of large peptides and proteins. The fruits of this endeavor, an understanding of how these macromolecules work, and therein, a basis for design of new structures that ultimately may be produced directly or by recombinant DNA methods, have begun to emerge.
