Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Abstract

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Discovery of Targeted Material Binding Microorganisms Using a Centrifugal Microfluidic Platform

“…Several variations of this approach exist, utilizing a variety of display libraries, such as phage display, [ 7–9 ] yeast display, [ 10,11 ] and bacterial display. [ 12–16 ] Bacterial display systems, typically created in Escherichia coli (E. coli) , have been extensively utilized due to the relative ease of manipulation, rapid growth rate, and direct cell surface peptide expression characteristics. In contrast to phage display, the genetic material is passed on directly to the next sorting round during cell regrowth, rather than requiring reinfection of bacterial cells.…”

The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries

“…Specificity for PLA, for instance over other plastics, can also be improved by incorporation of negative sorting steps in future studies. Strategies including negative sorting have previously been shown to provide specificity over structurally similar biological targets [5,17,23], and are expected to analogously aid in biopanning peptides towards inorganic materials.…”

“…Biopanning rounds consist of several steps: 1) incubating the target material with the naive library culture, 2) washing unbound bacteria from the material surface, 3) placing the washed material with bound library members into fresh media, 4) allowing the bound members to amplify (resulting in the Round 1 library), and 5) repeating steps 1-4 for two total panning rounds. The pB33-dsRed-eCPX3.0 library was prepared for panning as previously described [6,17,26] with modifications depicted in Fig. 3 for the current study.…”

“…1b and c) would not occur because the remainder of the eCPX scaffold would not be produced or shuttled to the outer membrane. This interaction can be monitored using Fluorescence-Activated Cell Sorting (FACS) [17]. Additionally, we incorporated inducible DsRed fluorescence into the vector cassette ( Fig.…”

Improved Microbial Fuel Cell Performance by Engineering E. coli for Enhanced Affinity to Gold

“…The MFCs were held at a constant load of 1 MΩ for 5 h before a variable resistance sweep was conducted from 30 to 0.3 MΩ with a logarithmic spacing and a 10 min dwell at each resistance to allow the system to equilibrate. On the eCPX scaffold, peptide expression level inside and outside of the MFCs was determined for those cells expressing P2X at the C-terminus by monitoring median fluorescence intensity and percent binding to a fluorescent tag with affinity for P2X, YPet-Mona (YPet), using fluorescence activated cell sorting (FACS) as previously described in detail [50].…”