Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library.

BACKGROUND: Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. METHODS: In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. RESULTS: We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. CONCLUSION: The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries.

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.

Recombinant production of anti-HIV protein, griffithsin, by auto-induction in a fermentor culture.

Griffithsin (GRFT) is a novel anti-HIV protein isolated from the red alga Griffithia sp. The potent anti-viral activity of GRFT against both laboratory and primary isolates of HIV at picomolar concentrations makes this protein an attractive candidate microbicide to prevent sexual transmission of HIV. Here, we describe the recombinant production and purification of a biologically active hexa-histidine-tagged GRFT (His-GRFT) from Escherichia coli. To facilitate a large-scale production of recombinant His-GRFT, we tested different expression conditions to optimize the expression in the cytoplasm of E. coli to increase the overall production of soluble His-GRFT. Attempts to express His-GRFT in shake flask cultures resulted in a modest yield of soluble His-GRFT, with a large accumulation of the protein in inclusion bodies. The use of a fermenter and of a rich, auto-inducing medium allowed the total amount of His-GRFT per liter to be increased by about 45-fold, with approximately 70% of the protein expressed in the soluble fraction. N-terminal sequencing and MALDI-TOF analyses of the recombinant His-GRFT confirmed that the initial methionine residue was cleaved off. Recombinant His-GRFT showed equivalent activity with natural GRFT, both in respect to gp120-binding characteristics as well as anti-HIV activity. Size-exclusion chromatography analysis showed that both native GRFT and recombinant His-GRFT existed as homodimers in solution. The expression system described in this work provides a basis for the mass production of GRFT to allow further studies of the protein and investigation of therapeutic and preventive strategies against HIV.