Putting biology to work for you:

In vitro (directed) evolution and other techniques

Phage display• Make a combinatorial library of genes of interest• Put genes into a vector so that each gene product is expressed on the surface of a bacteriophage

– Function encoded by each gene is on surface of phage– Gene is inside phage

• Take collection of phage and select those with desired properties (e.g., binding to something)• Similar methods: yeast display, bacterial display, ribosome display

See animation of phage display here: http://www.dyax.com/discovery/phagedisplay.html

Figure A-15In vitro selection to produce human monoclonal antibodies or

increase affinity of existing monoclonal antibody

Generate library of heavy and light chain variable regions using

spleen DNA. Or introduce random

mutations into variable regions

genes of a specific antibody.

Clone into a phage so that each phage

expresses one VH-VL surface

fusion protein.

Multiply phage display library in

bacteria, bind phage to surface

coated with antigen. Wash away unbound

phage.

Repeat procedure (multiply

recovered phage, bind to antigen,

wash away unbound phage)

for several cycles. Recover specific

high-affinity antigen binding VH-VL regions.

Phage Display schematic1

23

4 5 6

7

8

Phage display to select for sequence-specific DNA binding proteins

• Zinc finger proteins are modular.

• Each finger contains two anti-parallel -strands, an -helix, and a Zn atom.

• The -helix from each finger inserts into the major groove of DNA.

A General Strategy for Selecting High Affinity Zinc Finger Proteins for Diverse DNA Target Sites

Greisman et al., 1997, Science 275: 657

First use 3-finger/DNA crystal structure to determine important protein/DNA contacts

A General Strategy for Selecting High Affinity Zinc Finger Proteins for Diverse DNA Target Sites

Greisman et al., 1997, Science 275: 657

Originally bound to this sequence

RNA aptamers -- antibody-like properties

Aptamers have been made against small molecules, peptides, proteins, organelles, viruses, cells

Vitamin b12-binding RNA

aptamer

Aptamer database: http://aptamer.icmb.utexas.edu/index.php

Let the immune system make enzymes for you

Catalyst must bind more tightly to transition state than to products or reactants.

Catalytic Antibodies

• Raise antibodies against a transition state analog

• Screen hybridomas for antibodies that catalyze desired reaction

Wedemayer et al. (1997) Science 276, 1665-1669.

Catalytic antibody that hydrolyzes cocaineZhu et al., 2006, Structure 14: 205-216

Compound that wasinjected to raise

antibodies

Transition state analog that was

crystallized with Fab

(Nonpsychoactive products)

/ barrel enzymes evolved from a common ancestor

But this takes a long time…

10% of enzymes are barrels

Directed evolution

http://ocw.mit.edu/OcwWeb/Biology/7-344Spring-2008/CourseHome/index.htm

In vitro evolution of enzymes

• Enzymes evolved for functions inside a living organism, not for biotechnology

– Might need long-term stability– Might need activity in non-aqueous solutions

• Produce new enzymes using recombinant DNA technology, but don’t know how by rational design

• Use directed evolution

Directed evolution experiment

From Frances Arnold’s website: www.che.caltech.edu/groups/fha

Some considerations…

• Don’t start with random sequences because there are too many (20N)

• Instead start with lightly mutagenized gene (e.g., error-prone PCR) or high level of random mutations to small part of gene

Most mutations are destabilizing, so simply increasing protein stability can increase

mutational robustness

∆Gf

Marginally stable parent protein

∆Gf

stable unstable

Stabilized parent protein

These previously unacceptable mutations are now acceptable.

(critical threshold stability)

“Protein stability promotes evolvability.” JD Bloom, ST Labthavikul, CR Otey, and FH Arnold. Proc Natl Acad Sci, 103:5869-5874 (2006).

Evaluating stability

Two state unfolding transitionN <--> D

Monitor property of folded protein as function of increasing temperature

Transition midpoint (Tm)

Shows that class I MHC molecules require bound peptide for thermal stability

“Family” shuffling experiment

From Frances Arnold’s website: www.che.caltech.edu/groups/fha

From Frances Arnold’s website: www.che.caltech.edu/groups/fha

Sequence Space Structure Space

multiple sequences 1 structure

Protein Library Design

Mayo lab, Caltech

Sequence Space Structure Space

1 structure

Protein Fold Prediction versus Protein Design

1 sequence

Mayo lab, Caltech

Computational Protein Design:Rationale by the Numbers

Residues Sequences Mass 18 1023 Baseball 37 1048 Earth 42 1054 Sun 59 1077 Universe

Combinatorial Explosion

1 proteinp residues20 amino acid types20

p sequences

Mayo lab, Caltech

Optimization of Rotamers by Iterative Techniques (ORBIT)

Apply to protein fold stabilization, enzyme design

ComputationalProtein Design

RotamerLibraries

Methods

Applications

ProteinBackbones

CombinatorialOptimizationAlgorithms

Atom-BasedForcefields

NegativeDesign

Mayo lab, Caltech

De Novo Protein Design: Fully automated sequence selection

Dahiyat & Mayo, 1997, Science 278: 82-87

Comparison of original and designedprotein structuresTarget fold: Zif268 (a zinc finger)

Designed proteinZif268 Zn finger

Stability Based Design: Protein G

• Bacterial protein involved in host immune system evasion

• 56 amino acid domain

• Objective was to stabilize protein while preserving structure and function

• Design focused on 26 core and boundary positions

• Combinatorial complexity, 106 amino acid sequences

Mayo lab, Caltech

ON

H

O2NON

H

O2N

O-

C

O2N

N

O O O O HO O

catalytic antibody: kcat/kuncat = 106

Thorn et al., Nature 1995Debler et al., PNAS 2005

5-nitrobenzsoxazole

O O

ONN

H

O

O

General Base Asp/Glu

*

*

Phe/Trp

HO H-bond donorSer/Thr/Tyr

H N

N

H-bond donorHie/Hid/Gln/Asn

active site templateab initio t.s. model

Hu et al., JACS 2004

Kemp Elimination: a model system for enzyme design

Mayo lab, Caltech