directed evolution · directed evolution to allow expression of hrp in an active form in e.coli.!...

1© Dr. G. Gilardi, PEDD, Biological Sciences, Imperial College, London.

DIRECTED EVOLUTION


Introduction

! Knowledge of the sequence-structure-function relationships is not essential.

! Procedure:

! Requirements:! functional expression of the target enzyme in suitable microbial host

! availability of a screen/selection method

! identify a workable evolution strategy

! Steps in directed enzyme evolution:! Creation of a library of mutated genes

! selection/screening for genes with beneficial mutations

! cycles of mutation/selection/screen to accumulate beneficial mutations

CLONING

SCREEN/SELECT

MUTAGENESISLibrary of variants

EXPRESSION

cycles


A. Creation of a library of mutated genes

! Most frequently used methods:! 1. oligonucleotide-cassette mutagenesis! 2. error-prone PCR! 3. DNA-shuffling

! 1. Oligonucletide cassette mutagenesis! only for selected stretches of DNA! target AA in the same region of Iary sequence! combinatorial within the limits of the oligo.! labour intensive, not practical for many rounds of mutagenesis

! 2. Error-prone PCR:! Leung et al., 1989! low-fidelity polymerisation conditions, by varying conc. of Mn2+ and dNTPs! random mistakes are introduced


How to introduce errors in PCR:

! Taq:! highest known error rate, 0.1 x 10-4 - 2 x 10-4 per nucleotide

per pass of the polymerase;! increased to 5U (instead of 1U) to promote the chain

extension beyond the positions of base mismatch.

! MgCl2:! increased to 7 mM to stabilise non-complementary pairs;

! MnCl2:! added at 0.5 mM to diminish the template specificity of the

polymerase;

! dCTP and dTTP:! increased to 1mM to promote mis-incorporation (other at 0.2

mM).


! 3. DNA-shuffling:! higher upper limit (> 0.5-1.0 kb)

! allows for block changes (against gradual)

! procedure:

! collection of a pool of homogeneous mutated genes

! fragmentation by DNase I digestion (1kb to 10-50bp)

! reassemby by PCR (template switching = recomb.)

! selection

! cycles

! backcross with parental or wt DNA


Pool of homologous genesor

Library of random mutants of a parental gene

Random fragmentation

by DNase I

Pool of random DNAfragments

Reassembly

by PCR

Recombined DNA sequenceswith cross-over

Select of screen

best recombinant(s)Identify positive mutants

Eliminate negative mutations

Repeat


DNA-shuffling, Stemmer, 1994

Selection oncefotaxime

Note the cross-over (sexual PCR)


! Stemmer, 1994: directed evolution of ß-lactamase.

Note: for in vitro molecular evolution, recombination of blocks of sequence is more important thanpoint mutations alone.


! Recombination allows to combine two or moreproperties evolved separately:


Evolution:Local fitness landscapes

! Method to depict the extent to which anenzyme exhibits adesired characteristic


DNA shuffling

! Key features of DNA shuffling are:! the requirement of considerable quantities of DNA templates

with homologous regions able to prime each other;

! the presence of sequences of homology separated byregions of diversity;

! the block changes typical of sexual recombination broughtabout by template-switch or cross-over.

! These features have been subject of studies that led tomodifications and improvements of original basicprotocol.


Staggered Extension Protocol (StEP)

! Dramatically decreased the amount of theDNA template required by DNA shuffling.

! Allows the template switching during thesynthesis yields to chimeric genes withoutthe requirement of the DNase Ifragmentation step.

! Parental-like genes are eliminated bydigestion with Dpn I* as described for theQuikChange method.

* 5’-Gm6ATC-3’

Brief PCR

extension

Brief PCR

extension with

random priming

Repeat with

varying extension

Library of hybrid genes

StEP


Iteractive Truncation for the Creation ofHybrid Enzymes (ITCHY).

! Allows the generation of N- and C-terminal fragment libraries of twogenes by progressive truncation ofthe coding sequences withexonuclease III, followed byligation of the products to make asingle cross-over hybrid library.

! Gives cross-overs throughout allthe coding region. Only a smallnumber of cross-overs will connectthe two parent genes at siteswhere the sequences align.

Incremental

truncation

Ligation


ITCHY


Sequence Homology Independent ProteinRecombination (SHIPREC).

! Able to generate libraries of hybridgenes from distantly relatedsequences;

! It ensures cross-overs at positionssharing a similar structuralenvironment, maximising theprobability of obtaining functionalproteins.

! It creates a library of chimeric DNAsequences where the gene that wasat the 5’ position in the dimer will nowbe at the 3’ position and will donatethe C-terminus of the hybrid protein.The cross-over will therefore bedistributed over the entire length ofthe gene.

+Fusion

Fragmentation

by DNase I

Blunt-end

ligation

Size separation

Linearisation

digestion


SHIPREC


B. Selection and screening

! How many variants can we generate?

nv = 19 m [n!/(n-m)!m!]nv = number of variants; m = number of substitutions; n = number of AA


Selection and screening

! “You get what you screen/select for”

! Selection = microbial host:! Pre-requisite: function that confers a growth or survival advantage to the host organism

! Example: E.coli with TEM-1 ß-lactamase gene in presence of cefotaxime.

MIC increased from 0.01 !g/mL to 640 !g/mL (32,000-fold increase).

! Disadvantage: adaptation to selective pressure with alternative mechanism

! Screening = high-throughput assay:! Steps:

! construction of an arrayed protein library

! application of a high-throughput assay

! set sensitivity and specificity to identify positives


Screening

! Semi-quantitative visual screens:! In vitro colour screen strategies:

! cyt. b562: Brunet et al., 1993! red periplasmic extracts

! plastocyanin: Ybe et l., 1996! blue periplasmic extracts

! lactate dehydrogenase: Hawrani et al., 1996! lysis and immobilisation on nitrocellulose! enzyme activity with substrates/inhibitors/allosteric activators generated reduced

NAD+

! NADH coupled reactions involving phenazine methasulphate and nitrobluetetrazolium yield to a blue insoluble precipitate

! In vivo colour screen strategies:! subtilisin E: Arnold et al., 1996

! zone clearing or “halo” on casein/skim milk plates

! green fluorescent protein:! 104 - 105 clones visually screened for increased intensity of the intrinsic

fluorescence signal upon UV illumination


Screening

! Quantitative screen in 96-well plates:! quantitation and automation:

! robotic arms

! liquid handling systems

! plate readers, computerised data acquisition

! goals for directed evolution of enzyme activity:! increased thermostability

! increased stability and activity in artificial env.

! improved substrate specificity

! increased activity on novel substrates

! increased enantioselectivity

! example: development of a thermostable subtilisin E (1E2A)


Development of a thermostable subtilisin E (1E2A)


Subtilisin


Screening method on P450cam

! Mutants of P450 cam from Pseudomonas aeruginosa that are able to utilisethe electrons and oxygen provided by hydrogen peroxide, eliminating theneed of expensive cofactors and complex redox chains.

! P450 cam was evolved to acquire hydroxylating activity towards the un-natural substrate naphthalene.

! The screening procedure: co-expression of the horseradish peroxidasemutant Asn255Asp (HRP1A6). This mutant was previously obtained bydirected evolution to allow expression of HRP in an active form in E.coli.

! After 16h growth on agar/terrific broth plates, the P450 cam variants andHRP1A6 were replicated using a nitrocellulose membrane and transferredonto fresh agar plates containing 6 mM naphthalene and 10-30 mMhydrogen peroxide.

! The naphthols generated by the P450 cam variants were converted byHRP1A6 into fluorescent dimers and polymers that were detected byfluorescence digital imaging. Different naphthols obtained by diverse P450cam variants of altered regio-specificity of hydroxylation are polymerised byHRP1A6 in products with different fluorescence spectra.

! These characteristics were used to select P450 cam variants tuned tovarious regio-specificity of catalysis. Variants with improved properties weresubjected to in vitro recombination by StEP, yielding to several mutants with~ 20 fold improvement in naphthalene hydroxylation activity over the wildtype.

O

O

OH

naphthalene

naphthols

fluorescentdimers/polymers

Fluorescencedigital imaging

P450camH2O2

HRP1A6H2O2


Screening method on P450 BM3

! Wild type P450 BM3 hydroxylates fattyacids of chain length between C12 andC18.

! Arnold and co-workers engineered thisenzyme to hydroxylate short chain alkanessuch as octane.

! The screening procedure used an octaneanalogue, 8-pnpane, that upon terminalhydroxylation produces an unstablehemiacetal. This decomposes into thealdehyde and p-nitrophenolate that isdetected by its yellow clour at 410 nm

! Two round of error prone PCR and thescreening of 2,000-3,000 colonies produceda variant with activity 2-3 folds higher thanthe wild type.

unstable hemiacetale

NO2

O

NO2

O

HO

NO2

OH

O

H

+

P450 BM3

NADPH + O2

8-pnpane

p-nitrophenolate(yellow)

Absorbance at 410 nm


Screening method on NAD(P)H-dep. redoxenzymes, like P450 BM3

! A general screening procedure developed usingP450 BM3, but applicable to all NAD(P)H-dependent enzymes

! The screening procedure relies on the conversion ofthe NADP+ resulting from enzymatic activity into afluorescent product upon treatment with alkali

! The procedure can be applied to purified enzyme aswell as to whole E.coli cells expressing the targetNAD(P)H-dependent enzyme.

! The wide applicability, the use of whole cells, thesimplicity of procedure, the suitability for a microtiterplate format are very important properties for highthroughput screening purposes..

NAD(P)H

NAD(P)+

1 2

SUBSTR.

PRODUCT

Fluorescent product

Alkali assay

Enzyme

Absorbance at 360 nmFluorescence emissionat 460 nm


Back-up plateScreen plate(s)

Add substrate(s) + NAD(P)H

Activity test Uncoupling test

Alkali product H2O2/HRP/ABTS A = 360 nm A = 410 nm

Identification of positive(s)

DNA sequencing

Cell growth

Example of screeningprocedure:

random mutants

Lauric acid

Caffeine

Clozapine

Debrisoquine

Ibuprofen

Propranolol

wt ctrl

0

0.05

0.1

0.15

300 350 400 450 500

Wavelength (nm)

Absorbance

300 350 400 450 500Wavelength (nm)

0.15

0.1

0.05

0

Abso

rbance

0

20

40

60

80

100

120

140

160

180

420 470 520 570

Wavelength (nm)420 470 520 570 Wavelength (nm)

180

140

100

60

20

Flu

or.

em

issi

on+ substrate

+ substrate

no substrateno substrate


Display and selection

! Key to success in combinatorial biology procedures:! efficiently display the library! efficiently select the library

! Display procedures:! display on phages! display on bacteria! display on viruses and yeast

! Phage display:! variety of polypeptides, of different sizes and nature:

! small structured peptides;! .cytokines, such as human growth hormone, interleukin-3;! enzymes, such as ß-lactamase;! .receptors, such as CD4;! antibody fragments, Fab and scFv;! DNA-binding proteins.




Schematic models of an antibody molecule and a Fab fragment.



The phage M13

! M13 filamentous phage infectsgram negative bacteria (E.coli)

containing the F coniugativeplasmid;

Negatively stained micrograph Structural organisation of M13 of the virion


Life cycle and genome

! Life cycle in E.coli:! infection; synthesis of RFDNA; assembly

! Genome:!replication: genes II, V, X!assembly: genes I, IV, XI!Capside: genes III, VI, VIII, IX


Fusion vectors

! Simple fusion:! type 3! type 8

! Helper phage:! type 3+3

! 0-5 copies

! type 8+8! 0-2800 copies

! Double copies:! type 33! type 88


How are fusions accommodated?

! Gly-Gln-Ala-Ser-Gly was inserted between 4 and 5 aa position of gp8.

! X-ray diffraction of oriented M13 fibres at 7Å resolution:

WT M13 M13 fused with GQASG Difference

! The peptide was in an extended conformation in a shallow groove between 2alpha helices of gp8.

! The extended conformation is important for the interactions.


Applications of phage display

! George Smith, 1985:! EcoRI endonuclease on gp3

! selection on polyclonal antibody! 1,000 fold enrichement of fusions

! Two important concepts in phage display:! libraries of up to 108 clones can be built! phenotype-genotype are physically linked

! Applications to natural peptides:! mapping epitopes:

! fragments of DNA encoding portions of antigens fused with gp3. Selectionagainst immobilised antibodies. Applied for the gag gene of HIV.

! generating immunogens:! fragments of proteins are displayed to elicit antibodies against the coat protein

of parasites and viruses. Applied to the circumsporozoite protein of humanmalaria Plasmodium falciparum, with production of relevant Ab.

Also applied to variuos HIV coat proteins.


! Applications to syntheticpeptides:! Random peptide libraries are

generated by syntheticoligonucleotides of equal lengthbut random sequence

! Affinity based selection is calledbiopanning

! Phages are recovered, amplifiedand selected again bybiopanning.

! Several round of selection areusually carried out


! Random peptide libraries can be used for severalpurposes:! Antibody engineering

! Many receptors (SH3)


Applications to proteins:


! Among the many application areas:! Antibody engineering [*]

! Protein folding

! Enzyme catalysis! Catalytic antibodies [*]

! ß-lactamase [*]

! Subtilisin [*]

! DNA-binding proteins [*]

! DNA-binding proteins displayed on phages.! The problem: lack of unique correspondence protein amino acid - DNA base.

! It follows that multiple amino acid changes are required to alter DNA-binding specificity.

! A successful approach: phage display of zinc fingers.


Phage display of zinc fingers

! Zinc finger proteins: multiples of modules (fingers) that recognises 3bp;! Engineering modules for all 64 possible codons, means recognise any DNA sequence.! Klug and cowork.:

! 3-finger sequence,

Zif268, recogn. 9bp! randomised;! hybridisation with biotinylated-DNA! immobilisation with streptavidin! enzyme-linked

immunosorb. assay! strength of binding gray-scale, prop. enzyme activity! database reading! Kd calculation


Approaches.

EXPRESSION

RATIONAL DESIGN

SITE-DIRECTED MUTAGENESIS

STRUCTURE-BASED DESIGN

Analysis of 3D structures/models

STRUCTURAL DETERMINATION

DNA SEQUENCING

FUNCTIONAL CHARACTERISATION

STRUCTURAL DETERMINATION

RANDOM MUTAGENESIS

Error prone PCR / Homologous gene family

RECOMBINATION

DNA shuffling

FUNCTIONAL SCREENING OF LIBRARY

Selection (in vivo) / Screening (in vitro)

DNA SEQUENCING

FUNCTIONAL CHARACTERISATION

DIRECTED EVOLUTION


Combinatorial biology

! Combinatorial biochemistry is older than combinatorial chemistry.! Procedure.

! Preparation of the library:! Merrifield synthesis of peptides! DNA recombinant methods

! cassette mutagenesis! random PCR! DNA shuffling

! Display and screening:! Phage display

! phage M13 structure, cycle and genome! fusion vectors! applications

! antibodies! DNA-binding proteins

! Display on bacteria! Display on viruses and yeast


Concluding

! Concluding remarks:

! The development of combinatorial methods has radically changed the approach to design

! Only few libraries are able to immediately yield to molecules with the desired properties.! The design of initial as well as derivative libraries owes much to information acquired in rational design.! The refinement cycles also resemble those in rational design, where successive modifications are based

on new information acquired in previous cycles. For example, a lead isolated from a peptide librarycontaining ß-turn motifs constrained between disulphide bonds, can be subsequently modified by thecreation of derivative libraries where the diversity in amino acids is compatible with known structuralmotifs.

! References:

! Phage display of peptides and proteins. Brian K K, Winter J, McCafferty J: 1996, Academic Press, SanDiego:1-38

! Selection of DNA binding sites for zinc fingers usign rationally randomised DNA reveals codedinteractions. Choo Y, Klug A: PNAS 1994, 91 11168-11172.

! Structure of a foreign peptide displayed on the surface of bacteriophage M13. Kishchenko G, Batliwala H,Makowski L: J. Mol. Biol. 1994, 241 208-213.

! Phage display, protein engineering by directed evolution. O'Neil K T, Hoess R H: Curr. Op. Str. Biol.1995, 5 443-449.

! Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.Smith G P: Science 1985, 228 1315-1316.


Directed evolution: Bibliography

! Kuchner, O., and Arnold, F. H. (1997). Directed evolution of enzyme catalysis. TIBTECH, 15, 523-530

! Stemmer, W. P. C. (1994). DNA shuffling by random fragmentation and reassembly: In vitrorecombination for molecular evolution. Proc. Natl. Acad. Sci. USA, 91, 10747-10751

! Stemmer, W. P. C. (1994). Rapid evolution of a protein in vitro by DNA shuffling. Nature, 370, 389-391

! You, L., and Arnold, F. H. (1994). Directed evolution of subtilisin E in Bacillus subtilis to enhance totalactivity in aqueous dimethylformamide. Prot. Eng., 9(1), 77-83

! Zhao, H., and Arnold, F. H. (1997). Combinatorial protein design: strategies for screening protein libraries.Curr. Opinion in Struct. Biol., 7, 480-485

! Zhao, H. M., Giver, L., Shao, Z. X., Affholter, J. A. and Arnold, F. H. (1998). Nature Biotechnology 16,258-261.

! Ostermeier, M., Shim, J. H. and Benkovic, S. J. (1999). Nature Biotechnology 17, 1205-1209.

! Sieber, V., Martinez, C. A. and Arnold, F. H. (2001). Nature Biotechnology 19, 456-460.

! Joo, H., Lin, Z. L. and Arnold, F. H. (1999). Nature 399, 670-673.

! Farinas, E. T., Schwaneberg, U., Glieder, A. and Arnold, F. H. (2001). Advanced Synthesis & Catalysis

343, 601-606.

! Tsotsou, G. E., Cass, A. E. G. and Gilardi, G. (2002) Biosensors & Bioelectronics 17, 119-131.

directed evolution · directed evolution to allow expression of hrp in an active form in e.coli.!...

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