Function-based isolation of novel enzymes from a large library

Abstract

Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of protein variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent dye (2) a positively charged moiety (3) the target scissile bond, and (4) a fluorescence resonance energy transfer (FRET) quenching partner. Enzymatic cleavage of the scissile bond results in release of the FRET quenching partner while the fluorescent product is retained on the cell surface, allowing isolation of catalytically active clones by fluorescence-activated cell sorting (FACS). Using a synthetic substrate with these characteristics, we enriched Escherichia coli expressing the serine protease OmpT from cells expressing an inactive OmpT variant by over 5,000-fold in a single round. Screening a library of 6 x 10(5) random OmpT variants by FACS using a FRET peptide substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as much as 60-fold. This approach represents a potentially widely applicable method for high-throughput screening of large libraries on the basis of catalytic turnover.

Capsular Antigen Fraction 1 and Pla Modulate the Susceptibility of Yersinia pestis to Pulmonary Antimicrobial Peptides Such as Cathelicidin

“…OmpT and PgtE cleave preferentially between two basic amino acid residues (14,17), explaining why CAMPs such as cathelicidins, with their repeated pairs of Arg and/or Lys residues, are optimal targets for these omptins (24,59). The efficiency (k cat /K m ) of OmpT for a peptide with an Arg2Arg cleavage site was fourfold better than that for a similar peptide with an Arg2Val cleavage site (42,65). Our results identifying the FIG.…”

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

“…Proteins were isolated as previously described, with minor modifications (20), to a final purity Ͼ90% as determined by SDS͞PAGE. For kinetic analyses, 10-20 nM of the purified enzymes were incubated with 20 M to 1 mM of the appropriate substrate in 0.1 M Tris͞10 mM EDTA (pH 8.0) at room temperature (25°C), and the reaction was monitored by HPLC on a Phenomenex C18 reverse-phase column, using the following gradient: 5% AcN͞95% H 2 O for 1 min, increasing to 95% AcN͞5% H 2 O over a period of 29 min and returning to 5% AcN͞95% H 2 O over 5 min.…”

“…With these considerations in mind, we have extended our previous approach (20) and developed a new two-pronged strategy in which catalytic activities over a wide dynamic range for both a selection substrate and one or more counterselection substrates are quantified simultaneously at the single-cell level, enabling the rapid screening of mutant libraries. This approach relies on fluorescent substrates of different colors that label the surface of E. coli cells upon cleavage by a surface-anchored enzyme (20).…”

“…This approach relies on fluorescent substrates of different colors that label the surface of E. coli cells upon cleavage by a surface-anchored enzyme (20). Enzymes can be displayed on the surface of Gram-negative bacteria by established techniques (21,22), and thus, access to the fluorescent substrate is assured.…”

Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli -expressed libraries

“…The scFv-binding site exhibiting the highest affinity for PA has been humanized and converted to full-length IgG, and its neutralizing potential to anthrax intoxication is being evaluated in preclinical studies. In addition to affinity maturation, APEx can be exploited for several other protein engineering applications, including the selection of enzyme variants with enhanced function, because the cell envelope provides sites for retention of enzymatic catalytic products, thereby enabling selection based directly on catalytic turnover (38), or for the analysis of membrane protein topology, whereby a scFv antibody anchored in a periplasmic loop is able to bind fluorescent antigen and serves as a fluorescent reporter.…”