scholarly journals Directed Evolution of a Cp*Rh III ‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification

ChemBioChem ◽  
2020 ◽  
Author(s):  
Shunsuke Kato ◽  
Akira Onoda ◽  
Naomasa Taniguchi ◽  
Ulrich Schwaneberg ◽  
Takashi Hayashi
Keyword(s):  
Directed Evolution ◽  
Affinity Purification ◽  
Screening Platform

scholarly journals Cover Feature: Directed Evolution of a Cp*Rh III ‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification (ChemBioChem 4/2021)

ChemBioChem ◽  
2021 ◽  
Vol 22 (4) ◽  
pp. 593-593
Author(s):  
Shunsuke Kato ◽  
Akira Onoda ◽  
Naomasa Taniguchi ◽  
Ulrich Schwaneberg ◽  
Takashi Hayashi
Keyword(s):  
Directed Evolution ◽  
Affinity Purification ◽  
Screening Platform

scholarly journals Fluorescent Assay for Directed Evolution of Perhydrolases

2012 ◽  
Vol 17 (6) ◽  
pp. 796-805 ◽  
Author(s):  
Dragana Despotovic ◽  
Ljubica Vojcic ◽  
Radivoje Prodanovic ◽  
Ronny Martinez ◽  
Karl-Heinz Maurer ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput Screening ◽  
Saturation Mutagenesis ◽  
Fluorescent Assay ◽  
Assay Sensitivity ◽  
Subtilisin Carlsberg ◽  
Microtiter Plates ◽  
Wide Ph Range ◽  
Screening Platform ◽  
Ph Range

Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the µM range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4′-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5–9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased kcat for perhydrolytic activity compared with wild type.

scholarly journals A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool

2016 ◽  
Vol 52 (75) ◽  
pp. 11239-11242 ◽  
Author(s):  
Xuejun Zhu ◽  
Peyton Shieh ◽  
Michael Su ◽  
Carolyn R. Bertozzi ◽  
Wenjun Zhang
Keyword(s):  
Directed Evolution ◽  
E Coli ◽  
Membrane Bound ◽  
Screening Platform

A fluorogenic screening platform enabled the engineering of a membrane-bound bifunctional desaturase/acetylenase for improved activity in E. coli.

scholarly journals Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins

2020 ◽  
Vol 48 (11) ◽  
pp. e63-e63 ◽  
Author(s):  
Laurens Lindenburg ◽  
Tuomas Huovinen ◽  
Kayleigh van de Wiel ◽  
Michael Herger ◽  
Michael R Snaith ◽  
...  
Keyword(s):  
Amino Acid ◽  
Directed Evolution ◽  
Functional Screening ◽  
Library Synthesis ◽  
Oil Emulsion ◽  
Site Saturation ◽  
Emulsion Droplets ◽  
Dna Libraries ◽  
Screening Platform ◽  
Split And Mix

Abstract Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method—termed SpliMLiB for Split-and-Mix Library on Beads—was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.

scholarly journals Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs

2014 ◽  
Vol 27 (10) ◽  
pp. 399-403 ◽  
Author(s):  
J.N. Copp ◽  
E.M. Williams ◽  
M.H. Rich ◽  
A.V. Patterson ◽  
J.B. Smaill ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Platform

scholarly journals A Phosphite-Based Sscreening Platform for Identification of Enzymes Favoring Nonnatural Cofactors

2021 ◽  
Author(s):  
Yuxue Liu ◽  
Zhuoya Li ◽  
Xiaojia Guo ◽  
Xueying Wang ◽  
Zongbao K. Zhao
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Directed Evolution ◽  
Malic Enzyme ◽  
Chemical Biology ◽  
Culture Media ◽  
Chemical Space ◽  
Per Se ◽  
Screening Platform ◽  
Phosphite Dehydrogenase

Abstract BackgroundEnzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants.ResultsThe growth-based selection platform was designed by coupling with nonnatural cofactor-dependent phosphite dehydrogenase (Pdh) mediated the conversion of non-metabolizable phosphite into phosphate in the culture media. Thus, Pdh variant that strongly favors nicotinamide cytosine dinucleotide (NCD), a NAD analogue, the feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli.ConclusionsHere, we built a phosphite-based screening platform for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Isolation of Multiple Types of Plasminogen Activator Inhibitors from Vascular Smooth Muscle Cells

1989 ◽  
Vol 61 (03) ◽  
pp. 517-521 ◽  
Author(s):  
Walter E Laug ◽  
Ruedi Aebersold ◽  
Ambrose Jong ◽  
Willian Rideout ◽  
Barbara L Bergman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Affinity Purification ◽  
Muscle Cells ◽  
Exchange Chromatography ◽  
Natural Resistance ◽  
Protease Nexin ◽  
Pai 1

SummaryLarge arteries have a natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors. Vascular smooth muscle cells (VSMC) representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture. These cells were found to produce large amounts of inhibitors of plasminogen activators (PA). Fractionation of VSMC-conditioned medium by heparin-affigel chromatography separated three immunologically and functionally distinct PA inhibitors (PAI), namely PAI-1, PAI-2 and protease-nexin I. The three inhibitors were characterized by functional assays and immunoblotting. PA inhibitor 2 (PAI-2) had little affinity for heparin, whereas PA inhibitor 1 (PAI-l) bound to heparin and was eluted from the column at NaCl concentrations of 0. 1 to 0.35 M. Protease-nexin I, eluted at NaCl concentrations of 0.5 M and higher. Most of the PAI-1 was present in the latent, inactive form. PAI-1 was further purified by ion exchange chromatography on a Mono-Q column. Partial sequencing of the purified PAI-1 confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI-1. Thus, human VSMC produce all three presently known PAI and these can be separated in a single heparin affinity purification step.

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