The use of light-directed combinatorial peptide synthesis in epitope mapping.

The application of light-directed combinatorial peptide synthesis to epitope mapping is described. Photolithography and solid phase peptide synthesis were combined in an automated fashion to assemble arrays containing 1024 peptide sequences on a glass support in ten steps with the precise location of each peptide known. The simultaneous synthesis of two slides containing three arrays of peptides each allowed for the independent screening of both a monoclonal antibody (mAb) and its Fab fragment at two different concentrations.

Improvement of production, assay and purification of streptavidin.

The production of streptavidin by Streptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay.

Human macrophage colony-stimulating factor heterogeneity results from alternative mRNA splicing, differential glycosylation, and proteolytic processing.

The single gene for human macrophage colony-stimulating factor (M-CSF, or CSF-1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M-CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines.

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells.

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1.

A water-soluble, monitorable peptide and protein crosslinking agent.

A novel, freely water-soluble, heterobifunctional crosslinking reagent, N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitro-4-benzenesulfonic acid (mal-sac-HNSA), was synthesized and used for conjugation of sulfhydryl (cysteine)-containing peptides to carrier proteins. Reaction with amino groups releases the dianion phenolate, HNSA, which allows convenient spectrophotometric quantitation of the reaction in progress. Since mal-sac-HNSA is completely water soluble, its concentration can be adjusted to maximize the rate of amine reaction and to minimize hydrolysis.

Nonisotopic M13 probes for detecting the beta-globin gene: application to diagnosis of sickle cell anemia.

M13 DNA probes labeled with biotinylated psoralen and a streptavidin-horseradish peroxidase conjugate provide nonradioactive detection of the sickle cell and normal alleles of the beta-globin locus. The two biotinylated probes contain single-stranded sequences complementary to two different Sau3AI restriction fragments from the 5' region of the beta-globin gene and double-stranded M13 vector sequences. These probes are labeled with biotinylated psoralen photochemically linked to DNA.

Detection of activated Mr 21,000 protein, the product of ras oncogenes, using antibodies with specificity for amino acid 12.

Antisera raised to a set of chemically synthesized peptides spanning position 12 of ras Mr 21,000 protein (p21) (residues 5 to 17) were able to distinguish between different forms of p21 according to the amino acid at the twelfth codon. The peptide immunogens differed in one amino acid corresponding to position 12 of the protein; the substitutions were valine, serine, arginine, aspartate, alanine, or cysteine at this position. Normal p21 contains glycine at position 12; the other amino acid substitutions are those which would result from a single base change in codon 12 and may therefore be the activating mutations most likely to occur in human tumors.

Hepatic synthesis of canavaninosuccinate from ureidohomoserine and aspartate, and its conversion to guanidinosuccinate.

This study continues the exploration of the mechanism for the formation of guanidinoacetate and guanidinosuccinate in the human [Clin. Chem. 21, 235 (1975)].