Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening.

Optimization of heterologous protein production in Escherichia coli.

Escherichia coli has long been the primary prokaryotic host for the synthesis of heterologous proteins. Recent advances have been made in the expression of complex proteins as soluble, functional molecules, complete with prosthetic groups, disulfide bonds, and quaternary structure. The development of alternative promoter and induction strategies has improved the options available for manipulating the expression conditions, which are frequently critical to soluble yield.

Analysis of novel streptavidin-binding peptides, identified using a phage display library, shows that amino acids external to a perfectly conserved consensus sequence and to the presented peptides contribute to binding.

Streptavidin-binding peptides containing the consensus amino acid sequence motif EPDW were identified using a phage display library. Phage presenting peptides containing these sequences bound streptavidin in a biotin-sensitive fashion and could be eluted with biotin. The previously identified 'streptag' peptide sequence (AWRHPQGG) competed with phage presenting the EPDW consensus sequence for streptavidin binding.

Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody.

Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteine-constrained peptides.