A Novel Helper Phage Enabling Construction of Genome-Scale ORF-Enriched Phage Display Libraries

Abstract

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Improved recovery of bacteriophage M13 using an ATPS‐based bioprocess

“…M13 was propagated in E. coli strain ER2738 grown in a 1 L Erlenmeyer flask (25% working volume) using Super Broth (SB) medium (30 g L −1 tryptone enzymatic digest from casein, 20 g L −1 yeast extract and 10 g L −1 MOPS) containing 70 µg mL −1 kanamycin. Briefly, the bacterial culture with an optical density at 600 nm of 0.5 was infected with bacteriophage M13 at a multiplicity of infection (MOI) of 22.5 . The culture conditions were 37°C, 250 rpm, and 16 h (Shaking incubator 1585, VWR, International, USA).…”

“…Briefly, the bacterial culture with an optical density at 600 nm of 0.5 was infected with bacteriophage M13 at a multiplicity of infection (MOI) of 22.5. 21 The culture conditions were 378C, 250 rpm, and 16 h (Shaking incubator 1585, VWR, International, USA). The culture broth obtained at this point was termed as crude extract which was characterized by the presence of M13, contaminant proteins and other constituents of E. coli as well as whole cells and cell debris.…”

“…ATPS were prepared on a fixed mass basis. Predetermined quantities of stock solutions were mixed with Milli Q water and mixed with an established quantity of pre-purified M13 feedstock (10 12 -10 13 pfu mL 21 for evaluation of V R and addition of sodium chloride).…”

“…The plates were allowed to cool and incubated overnight at 378C. Plaques formed were counted and the concentration of phage was determined as pfu mL 21 . The number of phage particles was calculated by multiplying the concentration of the sample by its respective volume.…”

Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

“…The entire ligation reaction was electroporated in E.coli TOP10F’ cells (4 µl ligation mix/100 µl cells). This produced CO1 cells (K) as described earlier [17]. The library sizes have been described in the methods.…”