Table 1.

Strengths and limitations of recombinant peptide chips

Strengths of recombinant peptide chips

• Peptide libraries are created via recombinational cloning (low frequency of background colonies; maintenance of pre-defined orientation/reading frame; high cloning efficiency). • Design ranges from entirely random peptide collections to customized content (soft randomization; scaffold-based). • Peptide lengths are not limited as is still the case for state-of-the-art chemical on-chip synthesis, particle-based printing or chemically pre-manufactured peptide spotting. • Growth, induction and lysis of library-transformed E. coli clones all takes place on a single nitrocellulose membrane. • Evaporation or merging of spots is not a major concern. • The technique is conceptually facile, robust, cost-effective, sensitive, and easily (up-)scalable; fast turnaround times. • Has many promising applications, e.g.: epitope mapping, alanine substitutions, replacement studies, truncation scans, positional/ scrambled peptide library screening, along with unbiased examinations without any a priori knowledge. • Limited throughput can be compensated by massive parallelization (applying e.g. elaborate pooling schemes). • Extracted hits are immediately available as clones. • Integration of controls for quality estimation, affinity assessment, and inter-blot normalization is possible. • ‘Cell-free’ nature increases the chance of confirming hits with usual methods (FP, SPRI, Western Blot, Co-IP, etc.).

Limitations of recombinant peptide chips

• At present reachable density (resolution) represents an only sparse sampling of the theoretically possible combinatorial random (nonamer/hexamer) peptide library diversity. • Number of peptides that can be screened in a single approach is several orders smaller than feasible in typical yeast two-hybrid (Y2H) or phage display settings. • Technical equipment for robotic clone picking, reagent dispensing and/or clone arraying (printing) might be needed (depending on the desired throughput rate). • Currently only evaluated for moderate and higher affinity (strong) binding strengths, not for weak interactors. • Incorporation of modified or non-proteinogenic (synthetic) segments during chip compilation is not possible.