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Background: Phage display technology has been established as a powerful screening approach to select ligands or peptides for binding to proteins. Despite rapid growth in the field, there has been a relative dearth of quantitative criteria to measure the effectiveness of the process of phage display screening. Since human serum albumin (HSA) has been extensively studied as a drug carrier to extend the plasma half-life of protein therapeutics, the use of phage display technology is required for identifying albumin-binding peptides as the very promising strategy of albumin-binding against albumin fusion. The construction of albumin-binding drug requires the assessment of a large quantity of HSA-binding peptide (HSA binder) candidates for conjugation with therapeutic proteins. The use of the linear epitope mapping method has allowed researchers to discover many HSA-binding peptides. However, it may be inefficient to select these peptides based on sequence identity via randomly sequencing individual phage clones from enrichment pools., Method: Here, a simple assessment method to facilitate phage display selection of HSA-binding peptides was recommended. With experimentally determined phage titer, one can calculate the specificity ratios, the recovery yields and the relative dissociation constants, which are defined as quantitative criteria for panning and characterization of the binding phage fused peptides., Results: Consequently, this approach may not only enable more rapid and low-cost phage display screening, but also efficiently reduce pseudo-positive phages selected as HSA binders for conjugation with therapeutic proteins., Competing Interests: The author declares there are no competing interests., (©2023 Shi.)

Abstract: Penicillin G acylase (EC 3.5.1.11) is 86-kDa large heterodimeric protein comprising two peptide A 23-kDa and peptide B 62-kDa, produced by intein-mediated auto-splicing of a 92-kDa precursor. Since penicillin G acylase was potentially employed in the preparation of a wide range of semi-synthetic β-lactam antibiotics from acyl side-chain precursors and β-lactam nucleus, directed evolution of penicillin acylase using phage display technology for extending its novel specificity is an interesting topic both of industry and academic. We fused the penicillin acylase to fd phage coat protein III and used pIII secretion signal sequence instead of penicillin acylase, which coupled gene and enzyme on phage particle and will be useful for directed evolution of penicillin acylase. Western blotting and enzyme activity assay were performed to demonstrate penicillin acylase has been functionally displayed on phage surface. Owing to the intimate association of enzyme activity and precursor processing in penicillin acylase, alterations of protein residues to make a phage library should be careful not to lead to processing defects. By site-directed mutagenesis, we have then identified effect of Ser B1 and Asn B241 variants on post-translational maturation of phage fused penicillin acylase. [Copyright &y& Elsevier]