We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistancetransducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene HI. A library of 3 x 108 recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of (3-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking frmding is that all 51 contained tyrosine as the Nterminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 FAM (Tyr-Gly-PheTrp-Gly-Met) to 8.3 ,#M (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7

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The filamentous phage coat protein plll has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of plll, adjacent to the signal sequence cleavage site. Many functionally defective pIl fusion proteins contained several positively charged amino acids in this region. These residues appear to inhibit proper insertion of plll into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles. Suppressor mutations in the priA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues. We conclude that insertion of pIlI fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism. The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.Filamentous bacteriophages have recently been used as a vehicle to display a variety of biologically active peptides and proteins. Libraries of peptides at or near the amino terminus of the bacteriophage adsorption protein, pIlI (13,16,43), and on the major coat protein, pVIII (19,24,30), have been created. pIl has also been used to express human growth hormone (5, 28) and active enzymes (11,32), and both pIlI and pVIII have been used to display antibody fragments (10,20,25,31). Because of biological constraints that might be imposed by the assembly and function of an infectious virus particle, not all peptides would be expected to be well expressed in the libraries. In this study, we examined the features of small peptides fused to plll that can limit their efficient display on virions, thus reducing the diversity of peptide libraries, and we investigated means to mitigate these constraints.The adsorption protein, pIll, has several functions, including termination of the virion during assembly, structural stabilization of the phage particle, and adsorption to the F pilus during infection of Escherichia coli (35). pIlI is translated as a precursor protein with an 18-amino-acid signal peptide which is removed by signal peptidase during assembly of the mature 42.5-kDa pIll into the inner membrane. Before virion assembly, the 39.4-kDa amino-terminal domain of pIII is projected into the periplasm, anchored to the membrane by a single membrane-spanning sequence near the C terminus. Fusion peptides in random libraries are located in the early mature region of pIll, near or immediately adjacent to the signal peptide cleavage site. The mechanism by which pIl inserts into the inner membrane is unknown, but the known incompatibility of certain peptide sequences with transport across the membrane (26) suggests that this is a step in which biological constraints might occur.E. coli membrane proteins whose assembly has been studied can be classified according to their dependence on the E. coli sec gene products. (49). Experi...

Crystallins are small heat shock proteins with chaperone function that prevent heat- and oxidative stress-induced aggregation of proteins. This is the first report describing modifications of alphaA crystallin in the sensory retina, including altered content and truncation with aging. Proteins from adult, middle age, and old Fischer 344 Brown Norway rats were compared. Western immunoblotting was used to evaluate alphaA crystallin content and identify protein spots on two-dimensional gels containing alphaA crystallin. The type and site of multiple post-translational modifications were identified by mass spectrometry. We found the content of alphaA crystallin was significantly decreased in the oldest rats. On two-dimensional gels, retinal crystallins resolved into multiple spots with altered migration, indicative of changes in intrinsic charge and/or truncation. Post-translational modifications that were identified included oxidation, phosphorylation, deamidation, acetylation, and truncation. In samples from rats of all ages, a highly modified N-terminus containing these modifications was found. We also observed an age-dependent difference in the extent of N- and C-terminal truncation. These results suggest that protection against stress-induced protein aggregation is compromised in the aged retina.

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