Browsing by Subject "Proteolytic enzymes"
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Item Engineering highly active and specific protease variants(2006) Varadarajan, Navin; Iverson, Brent L.; Georgiou, GeorgeThe reprogramming of enzyme catalytic activity and selectivity is a central issue in protein biochemistry and biotechnology. Numerous structure-guided and directed evolution strategies have been employed in search of enzyme variants that exhibit high catalytic rates with poor or inactive substrates of the parental enzyme. As impressive as these successes have been, the engineering of enzymes that exhibit turnover rates and selectivities with new substrates comparable to their natural counterparts has proven quite a challenge, especially when considering those enzymes for which a genetic selection strategy is not possible. By utilizing bacterial display and multi-parameter flow cytometry we have developed a novel methodology for emulating positive and negative selective pressure in vitro for the isolation of enzyme variants with reactivity for desired novel substrates, while simultaneously excluding those with reactivity towards undesired substrates. In order to demonstrate the application of the high-throughput flow-cytometric for protease engineering; we sought to evolve a set of highly active OmpT variants that have P1 specificities altered systematically to recognize one amino-acid from each of the six classes of amino acids By screening error-prone and multiple residue saturation libraries we describe the systematic directed evolution of a set of proteases with altered recognition sites. A set of OmpT variants were engineered that can specifically cleave substrates having a hydrophobic, polar, aromatic and even acidic residue at the P1 and Arg at the P1’. In particular we note that the change in electrostatic specificity from a basic amino acid (Arg) to an acidic (Glu) is unprecedented. After successfully changing P1 specificity, we then focused our attention on isolating OmpT mutants that recognize altered P1’ specificities such as Ala and Val. Towards this end, we show the isolation of highly active OmpT variants that cleave ArgVal and Glu-Ala sequences.Item Toward high throughput directed evolution of protease specificity using fluorescence activated cell sorting(2004) Gam, Jongsik; Whitman, Christian P.; Iverson, Brent L.Directed evolution by high throughput screening is used for improving protein properties and functions. In our laboratory, a FRET peptide probe was used with a Fluorescence Activated Cell Sorting (FACS)-based screening method for OmpT, a native outer membrane protease of E. coli. This earlier FRET probe enabled the isolation of an OmpT variant with altered specificity, although wild-type Arg-Arg cleavage activity was still present. Newly devised protease probes that operate via electrostatic capture on the bacterial cell surface allowed a very powerful “twocolor” type of FACS sorting, in which new catalytic activities are selected while simultaneously deselecting unwanted activities. Newly designed autoquenching peptide substrates with two identical fluorophores (BODIPY) were used for post screening of new variants on 96 well plates. Using the two-color FACS sorting approach, we were able to isolate OmpT variants 1.2.19 and 1.3.19, displaying a million-fold conversion of specificity (high Ala-Arg cleavage and very low wildtype Arg-Arg cleavage). This study demonstrates that the two-color sorting system for protein evolution can be used for pinpointing protein functions such as protease specificity and scFv antibody. To apply Periplasmic Expression with Cytometric Screening (PECS) for high throughput directed evolution of exogenous proteases, a permeabilization study of the E. coli outer membrane was carried out using FACS with cells expressing periplasmic scFv antibodies. Probes (>1000 Da) hardly gave practical signals for FACS sorting without a permeabilizer. However, outer membrane permeability toward a peptide probe (~ 2800 Da) was significantly enhanced by adding polymixin B nonapeptide (PMBN) or using a “heptoseless” LPS (lipopolysaccharide) E. coli mutant, D21f2. Based on the PECS system, the active form of chymotrypsin, potentially toxic to its hosting cell, was successfully expressed in the periplasm, and its activity was detected using a fluorescent probe and FACS. However, when positive and negative cells were mixed, the FACS signal was not specific to the positive cells. We believe this is due to enzyme leakage to the medium prior to substrate cleavage. Although further FACS screen assays are required to improve signal resolution of positive and negative cells, this work provides the foundation for high throughput directed evolution study of exogenous proteases.