Enrichment of Open Reading Frames Presented on Bacteriophage M13 Using Hyperphage

Abstract

The enrichment of open reading frames (ORFs) from large gene libraries and the presentation of the corresponding polypeptides on filamentous phage M13 (phage display) is frequently used to identify binding partners of unknown ORFs. In particular phage display is a valuable tool for the identification of pathogen-related antigens and a first step for the development of new diagnostics and therapeutics. Here, we introduce a significant improvement of phage-based ORF enrichment by using Hyperphage, a helperphage with a truncated gIII. The methods allow both the enrichment of ORFs from cDNA libraries and the display of the corresponding polypeptides on phage, thus combining ORF enrichment with a screening for binding in one step without any further subcloning steps. We demonstrated the benefits of the method by isolating the sequences encoding two predicted immunogenic epitopes of the outer membrane protein D encoding gene (ompD) of Salmonella typhimurium. Here, we showed that when using a mixture of three constructs with only one containing an ORF solely this correct construct could be reisolated in phage particles. Further; both epitopes were detected by enzyme-linked immunosorbent assay (ELISA), demonstrating correct translation of fusion proteins. Furthermore, the enrichment system was evaluated by the enrichment of ORFs from total cDNA of lymphocytes. Here, we could show that 60% of the phage contained ORFs, which is an increase of an order of magnitude compared with conventional phage expression system. Together these data show that the Hyperphage-based enrichment system significantly improves the enrichment of ORFs and directly allows the display of the corresponding polypeptide on bacteriophage M13.

A human scFv antibody generation pipeline for proteome research

“…In addition, Hyperphage enriches intact open reading frames (ORFs) during packaging (Hust et al, 2006;Kügler et al, 2008;Naseem et al, 2009), which may also help to enhance the quality of the antibody gene library. The sequencing of the Hyperphage packaged libraries showed no significant differences in the abundance of different subfamilies for VH and kappa compared to the cloned libraries.…”

SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli

“…Furthermore, scFvs will be produced with N-and C-fusion protein partners for different purposes, for example, Dübel [44] or Mølhøj et al [45]. The impact on antibody phage display was evaluated by the production of scFv phage in microtiter plates (MTPs) using two different helperphages, M13K07 for monovalent display and the Hyperphage for polyvalent display [18,38,46] and subsequent analysis by ELISA. First, the yields of phage particles were not affected by the choice of leader, either for M13K07 or Hyperphage.…”

“…Antibody phage production in a 30 mL scale was performed according to Hust et al [38]. For antibody phage production in polypropylene microtitre plates (MTPs) (Greiner bio one, Frickenhausen, Germany), MTP wells were filled with 200 mL 2xTY medium [36] + 100 mg/mL ampicillin + 100 mM glucose.…”

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

“…The diversity of the library was estimated to be 1.13 ϫ 10 8 clones. The library was packaged using a protocol adapted from that for Fab (antigen binding fragment) phage production in reference 22: 2.5 ϫ 10 11 Hyperphage particles (19,48,53) were used. The packaging of the library was tested by titration and immunoblotting according to reference 22.…”