Screening for improved enzymes. Intro Producing enzymes with novel properties directed evolution library finding desired mutant (most critical step) enzymatic.

Præsentationer af emnet: "Screening for improved enzymes. Intro Producing enzymes with novel properties directed evolution library finding desired mutant (most critical step) enzymatic."— Præsentationens transcript:

1 Screening for improved enzymes

2 Intro Producing enzymes with novel properties directed evolution library finding desired mutant (most critical step) enzymatic activity is assayed with a microtitre plate Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

3 Intro Experimental limitations impose significant restrictions on the number of mutants that can be screened designing screens optimisation Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

4 Validation of a screen Different ways to plot data Property vs. position of clone in a plate Descending Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

5 Validation of a screen Histogram Coefficient of variation CV = σ / μ *100% Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

6 Errors Observe a very high CV or skewed distribution often come from differences in cell density and protein expression level another important is poor design of activity assay Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

7 Screening Generally screen for two types of property absolute values (e.g. enzyme activity) relative values of two properties (e.g. substrate specificity and thermostability; ratio of absolute values) Important: High reproducibility Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

8 Analysis Sequencing most accurate method Fraction of inactive clones most convenient way to set a good mutation rate for directed evolution Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

9 Analysis When screening for two properties simultaneously: improved or decreased property in both improved properties in one and decrease in another (and vice versa) Kilde: Arnold, Chapter 8 (Evaluating a screen and analysis of mutant libraries)

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11 Basis for alle screening & selection metoder Link mellem genet, enzymet det koder for og produktet, der dannes grundet enzymaktiviteten Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

12 Low-medium throughput screens Detektering af enzymatisk aktivitet i kolonier på agarplader el. ”crude cell lysates” ved produktion af en fluorofor el. kromofor Screene op til 10 4 i løbet af nogle dage Har været effektive til isolering af enzymer med forbedrede egenskaber, men for at kunne isolere varianter med en dramatisk forandret fænotype kræves mere effektive metoder Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

13 HTS af enzymer med phage display Fordele: Phagen giver linket mellem gen og protein Display på overfladen giver uhindret adgang til substrat og ønskede reaktionsforhold Udfordringen er at få linket enzym med produkt Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

14 HTS af enzymer med phage display Selektion af katalytiske antibodies med peroxidase aktivitet. Tyramine oxideres af hydrogenperoxid i en proces katalyseret f peroxidase antibodies fremvist på phagen. Biotin-tyramine bindes irreversibelt med phenol- sidekæder på peroxidase antibody. Selektion via biotin- streptavidin interaktioner Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

15 HTS af enzymer med phage display Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

16 HTS af enzymer med phage display ”Catalytic elution” Til enzymer der er afhængige af co-factors Efter protein ekspression på phage-overfladen, fjernes al co-factor og de katalytisk inaktive phages bindes til en immobiliseret substrat. Co-factor tilsættes, og phages der fremviser et aktivt enzym elueres idet enzymet omdanner substratet til produkt. Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), H. Pedersen et al., ” A method for directed evolution and functional cloning of enzymes”, Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 10523–10528, September 1998

17 HTS af enzymer med cell display Har mange ting til fælles med phage display Brug af FRET substrat FRET-based enzyme screening. (a) The structure of the FRET substrate: Fl, BODIPY; Q, tetramethylrhodamine. (b) Binding of FRET substrateto the cell surface of E. coli cells displaying the outer membrane protein OmpT. The positively charged FRET substrate is attached to the negatively charged polysaccharides of the cell surface. (c) Upon enzymatic cleavage of the scissile bond, the FRET substrate displays Fl fluorescence, which is otherwise quenched by Q. Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), S. Becker et al., “Ultra-high- throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts”, Current Oppinion in Biotechnology 2004, 15:323-329

18 HTS af enzymer med cell display På microbeads med growth medium Danner en bead population med, gennemsnitligt, en enkelt celle per bead Beads immobiliseres og cellerne gror og danner microkolonier Equilibreres med et chromogenic el. fluorogenic substrat Beads screenes under mikroskop Digital imaging of color changes resulting from h-lactamase activity displayed by colonies immobilized on adsorbed beads: gradual color change following 1-, 10-, and 30-min incubation with 10 mM PenG). Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), A. Freeman, “Screening of Large Protein Libraries by the ‘‘Cell Immobilized on Adsorbed Bead’’ Approach”, Biotechnology & Bioengineering, vol. 86, no 2, 2004

19 In vitro compartmentalization (IVC) Water-in-oil emulsion -> danner mikroskopiske vandige compartments. Hver compartment indeholder i snit et enkelt gen og fungerer som en kunstig celle, der tillader transcription, translation og aktivitet af det resulterende protein, at foregå i compartment’et (droplet volume ~5 fL). Oliefasen forbliver inaktiv og begrænser diffusion mellem compartments. Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), O.J. Miller, “Directed evolution by in vitro compartmentalization”, Nature Methods, vol.3, no. 7, 561-570, 2006

20 IVC selections Strategi: Substrat (dvs. efter- følgende produkt), til den ønskede enzym aktivitet, fysisk linked til genet Hermed kan enzym- kodende gener isoleres i kraft af deres fastgørelse til produktet Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), O.J. Miller, “Directed evolution by in vitro compartmentalization”, Nature Methods, vol.3, no. 7, 561-570, 2006

21 IVC selections Microbeads i w/o emulsion Genet for en phosphotriesterase er immobiliseret på en microbead. Enzymet udtrykkes med et tag (T), hvor der på beaden sidder anti-tag antibodies, hvormed enzymet efter translation fastgøres på beaden. Efter translation, anden emulsion indeholdende et substrat med en caged-biotin Det aktive enzym kløver ester-substratet, og biotin bliver uncaged ved bestråling, og både substrat og produkt bindes til beaden (biotin- streptavidin) Emulsionen ødelægges, beadsene inkuberes med monoclonale antibodies, der detektere produktet Inkubering med fluorescein-labelled sekundære antibodies – analyseret med FACS Kilder: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes), S. Becker et al., “Ultra-high- throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts”, Current Oppinion in Biotechnology 2004, 15:323-329

22 FACS (Fluorescens-activated cell sorter) Analysere > 10 7 per time http://www.bio.davidson.edu/COURSES/GENOMICS/method/FACS.html

23 IVC Fordele: In vitro system -> tillader valg af substrat, produkt og reaktionsforhold der kan være inkompatible med in vivo systemer Flere emulsions steps => man kan bruge forskellige forhold for translation og selektion for catalysis Enkelt emulsion step -> muligt at ændre indeholdet i compartments efter translationen uden at ødelægge emulsionen Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)

24 w/o/w Fordele: Behøves intet ”fysisk” link mellem produkt og gen Kan analyseres med FACS Kilde: Reymond, Chapter 6 (HTS screening and selection of Enzyme-encoded genes)