A high-capacity alkaline phosphatase reporter system for the rapid analysis of specificity and relative affinity of peptides from phage-display libraries.

We describe a novel reporter enzyme cassette system which enables the analysis of large numbers of linear and cyclic peptides in terms of their binding to a specific target molecule. In this system, peptides selected for target binding from random peptide phage-display libraries are expressed as cloned fusion proteins with bacterial alkaline phosphatase. The binding specificity and relative affinity of each peptide-enzyme fusion protein is then evaluated in a target-specific ELISA.

Binding epitope of somatostatin defined by phage-displayed peptide libraries.

We have developed a versatile phagemid system to display peptides on the surface of M13 bacteriophage at a copy number which approaches monovalency. In this system, a phagemid encodes a peptide fused to the amino-terminus of the second domain (dII) of the minor coat protein pIII under control of the inducible lac promoter. The fusion protein is displayed in combination with several copies of wild-type pIII on the surface of phage.

Expression, purification, and characterization of the human squalene synthase: use of yeast and baculoviral systems.

We have cloned and utilized a cDNA corresponding to the human squalene synthase gene to generate active enzyme from yeast and baculoviral expression systems. Expression of human squalene synthase in yeast resulted in production of active enzyme in cellular lysates. The presence of the active human enzyme, however, was insufficient to rescue growth of spores defective in yeast squalene synthase function, suggesting that structural differences in the yeast and human enzymes may affect localization or folding of the protein.

Primate antibodies to components of the human immune system.

The feasibility to raise nonhuman primate antibodies against selected components of the human immune system was tested. The immunogens were whole cells (human T lymphocytes) or purified, recombinant human proteins (cytokines: TNF alpha or GM-CSF; soluble forms of cell surface antigens: sCD4 or sCD25). Significant immunizations, yielding functionally relevant antibodies, were readily achieved in rhesus monkeys, but, not surprisingly, may be less frequent in chimpanzees.