Unnatural Protein Unnatural Protein Engineering:Engineering:Biochemical and Medicinal Biochemical and Medicinal ApplicationsApplications

Youngha RyuYoungha Ryu

Department of Chemistry Department of Chemistry Texas Christian UniversityTexas Christian University

Methods of Protein Modification

• Chemical modification of the reactive side chains

• Total chemical synthesis

• Ligation of synthetic peptide to a

truncated protein

• In vitro transcription/translation

• Expression in living organisms

Protein biosynthesis machineryDNA

mRNA

acylated tRNA

peptide chain

ribosome

H2N

mRNA

tRNA aa~AMP

Aminoacyl-tRNA synthetase (aaRS)

R

NH2

O

O

R

NH2

O

O

EF-Tu

“Standard” Genetic Codes

PNAS 48, 1086 (1962)

Suppressor tRNAs

Nonsense Suppressors in E. coli

Suppressor Type Anticodon change tRNA Gene Efficiency

supE Amber CUG --> CUA tRNAGln glnV 0.8-20%

supP Amber CAA --> CUA tRNALeu leuX 30-100%

supD Amber CGA --> CUA tRNASer serU 6-54%

supU Amber CCA --> CUA tRNATrp trpT

supF Amber GUA --> CUA tRNATyr tyrT 11-100%

supZ Amber GUA --> CUA tRNATyr tyrU

supB Ochre UUG --> UUA tRNAGln glnU

supL Ochre UUU --> UUA tRNALys lysT

supN Ochre UUU --> UUA tRNALys lysV

supC Ochre GUA --> UUA tRNATyr tyrT

supM Ochre GUA --> UUA tRNATyr tyrU

glyT Opal UCC --> UCA tRNAGly glyT

trpT Opal CCA --> UCA tRNATrp trpT 0.1-30%

In vitro System

P. G. Schultz et al. Science 1989, 244, 182

Incorporation of unnatural amino acids into proteins in living organisms

• Efficient transport or biosynthesis of unnatural amino acids

• Unique codons (nonsense, four base, etc)

• tRNA/aminoacyl-tRNA synthetase pair that is orthogonal to the endogenous system

• Directed evolution of the aminoacyl-tRNA synthetase to selectively charge the orthogonal tRNA with an unnatural amino acid

An “Orthogonal” pair from M. jannaschii

• M. jannaschii tRNATyr is orthogonal to E. coli synthetases

• M. jannaschii TyrRS is orthogonal to E. coli tRNAs

• M. jannaschii TyrRS has minimal interaction with anticodon

• M. jannaschii TyrRS has no proofreading activity

AOH

CCAGGCCGCC

pCCGGCGG

C G G C C

G C U G G

U U G

A A CCGCCU

GCGGA

T AA

A

CU UC

GG

UA

UACAG

G

GA G

CU

CU A

G

A

3'

5'

G

CA

G U

Directed Evolution of Mj TyrRS

Selection strategy

Wang, Brock, Herberich & Schultz Science 292, 498, (2001)

Synthetases for Unnatural amino acids

H2NOH

OH2N

OH

O

O

Turner, Graziano, Spraggon & Schultz J. Am. Chem. Soc., 127, 14976 (2005) Proc. Natl. Acad. Sci., 103, 6483 (2006)

Expanded Genetic Code in E. coli

H2NOH

O

O

H2NOH

O

H2NOH

O

O

H2NOH

O

O

H2NOH

O

I

H2NOH

O

NH2

H2NOH

O

N3

H2NOH

O

O

H2NOH

O

CO2H

H2NOH

O

C

H2NOH

O

NO2

H2NOH

O

B(OH)2

H2NOH

O

O

H2NOH

O

OH

OH

H2NOH

O

NN

H2NOH

O

O

NO2

SO

O

O-

H2NOH

O

N

H2NOH

O

N

N

H2NOH

O

CF3

NN

H2NOH

O

OO OH

Sub-optimal yields of proteins in E. coli

• Non-versatile two plasmid system? – Integration of the synthetase and tRNA genes into a single plasmid, which is compatible with most E. coli expression vectors and strains

• Intrinsic low efficiency due to the competition with termination? – High suppression efficiency is achieved by naturally occuring non-sense suppressors (e.g. XL1-Blue)

• Inefficient transcription and processing of tRNA? – New promoter and flanking sequence

• Inefficient expression of aminoacyl-tRNA synthetase? – New promoter

New Mj tRNA expression cassette

ACCAGGCCGCC

ACCAGGCCGCC

CCGGCGG

CCGGCGG

C G G C C

G C U G G

U U G

A A CCGCCU

CGCCU

GCGGA

GCGGA

U

A A

A

CU UC

GG

UA

UACAG

GG

A G

C

UC U A

A

A

3'

5'

G

CA

GC

U

A A U U C G A A A A G C C U G C U C

U

A

A

CU U U U U C G G A C G A GU

RNAse P RNAse E

RNAse T, PH etc

RNAse III

JYTRN

Terminator

• E. coli prolyl tRNAs have C1-G72 pair, which is major identity determinant of MjtRNA – Important context for the precise tRNA processing

• proK tRNA is most frequently used in E. coli

• FIS enhances tRNA transcription

Mutant glnS promoter enhances the synthetase expression

AAAAAACTAACAGTTGTCATTGTCAGCCTGTCCCGCTTATAAGATCATACGCCGTTATACGTT

AAAAAACTAACAGTTGTCAGCCTGTCCCGCTT-TAATATCATACGCCGTTATACGTT

-35 region -10 region +1

WT

Mutant

BpaRS

WT Mutant

Asp286Arg (D286R) substitution enhances tRNA(CUA) binding affinity

Asp286

His283

G34

Phe261

Asp286 Arg286

G34C34 C34

TyrRS (WT) – tRNATyr (WT)

Asp286

TyrRS (WT) – tRNATyr (CUA) TyrRS (D286R) – tRNATyr (CUA)

Km= 0.35 Mkcat= 0.19 s-1

kcat/Km (relative) = 1

Km= 39 Mkcat= 0.070 s-1

kcat/Km (relative) = 0.0033

Km= 0.68 Mkcat= 0.079 s-1

kcat/Km (relative) = 0.22

Kobayashi et. al. Nat. Struct. Biol. 10, 425 (2003)

Polycistronic expression of MjtRNA

tRNA copy #

tRNA promoter

0 1 1 1 3 6

lpp proK proK proK proK

aaRS promoter glnS glnS’ glnS’ glnS’glnSglnS

-

JYTRN

-Galactosidase assay for suppression efficiency

araBAD promoter leader

TAG

lacZ

proK

proK + D286R

proK + glnS’

proK + glnS’ + D286R

+ 3TRN

+ 6TRN

Efficient incorporation of many different unnatural amino acids

H2N CO2H

O

H2N CO2H

O

H2N CO2H

N3

H2N CO2H

I

Bpa pAcPhe pAzPhe pIPhe

Efficiency and fidelity

Optimizing protein yields in E. coli

• E. coli prolyl-tRNA promoter and terminator for the amber suppressor tRNA

• Mutated form of the glnS promoter for the synthetase

• D286R substitution in the synthetase gene

• Multiple copies of the suppressor tRNA gene

• Yield of adiponectin (Glu123Bpa) mutant: 0.4g/L

Ryu & Schultz Nat. Methods 3, 263 (2006)

Site-selective modification of proteins

NH

O

O

H2NO PEG

NH

O

NO PEG

pAcPhe

NH

O

Lys

NH2

NH

O

HN

O

PEGO PEG

O

N

O

O

http://www.ambrx.com

Photocaged Tyrosine

Tyr503

lactose

H2N CO2H

O

O2N

H2N CO2H

OH

ONBY Tyrosine

h

365nm

Deiters, Groff, Ryu, Xie & Schultz Angew. Chem. Int. Ed. 45, 2728 (2006)

Incorporation of a distance probe into proteins

H2N CO2H

NO2

pNO2Phe

Tsao, Summerer, Ryu & Schultz J. Am. Chem. Soc. 128, 4572 (2006)

Incorporation of an IR probe into proteins

__O2

[2230 cm-1]

Cyanide [2236 cm-1]

NO [2230 cm-1]

Azide [2234 cm-1]

CO [2239 cm-1]

Met-ferric [2248 cm-1]

Deoxyferrous[2233 cm-1]

Ferric (Fe3+) adducts

Ferrous (Fe2+) adducts

Wavenumber (cm-1)

2160 2180 2200 2220 2240 2260

Re

lati

ve

Ab

so

rba

nc

e

0.0

0.2

0.4

0.6

0.8

1.0

FeII(CO) pCNPhe64 Mb

FeII(NO) pCNPhe64 Mb

FeII(O2) pCNPhe64 Mb

2230 2239B

H2N CO2H

CN

Schultz, Supekova, Ryu, Xie, Perera & Schultz J. Am. Chem. Soc. 128, 13984 (2006)

pCNPhe

Co-translational protein modification

H2N

OH

OH

O

Tyrosine

Desulfo-hirudine Sulfo-hirudine

Liu & Schultz, Nat. Biotech. 24, 1436 (2006)

Thrombin Thrombin

Ki = 307 fM Ki = 26 fM

H2N

O

OH

O

Sulfotyrosine

S

O

O

O-

Incorporation of an NMR probe

Cellitti et. al. J. Am. Chem. Soc. 130, 9268 (2008)TE domain of the human FAS

H2N CO2H

O F

FF

Unnatural amino acids incorporated by the mutant TyrRS in E. coli

H2NOH

O

O

H2NOH

O

H2NOH

O

O

H2NOH

O

O

H2NOH

O

I

H2NOH

O

NH2

H2NOH

O

N3

H2NOH

O

O

H2NOH

O

CO2H

H2NOH

O

C

H2NOH

O

NO2

H2NOH

O

B(OH)2

H2NOH

O

O

H2NOH

O

OH

OH

H2NOH

O

NN

H2NOH

O

O

NO2

SO

O

O-

H2NOH

O

N

H2NOH

O

N

N

H2NOH

O

CF3

NN

H2NOH

O

OO OH

Identification of the protein modification and secretion pathways by photo-crosslinking in E. coli

N-Acetylation of recombinant proteins in E. coli• N-Acetylation (e.g. Z-domain etc)

• N-Acetylation of lysine side chains (e.g. Porcine and bovine somatotropins)

Secretion pathway of the YebF protein in E. coli

NH

O

O NH

O

HO NH

R

O

Bpa

O

H

R

NH

h

N-Terminal acetylation of the Z-domain depends on E. coli strains and expression plasmids

N-Terminal acetylation of the Z-domainis context-dependent

Position Amino acidObserved

M.W.- Met1 N-Acetylation

S3Bpa 7968.3

Yes YesTyrosine 7886.5

V4Bpa 7913.9

Yes NoTyrosine 7827.4

D5Bpa 7898.2

Yes NoTyrosine 7811

Photo-crosslinking and proteomics analysis

Band S3Bpa V4Bpa D5Bpa

1 Protein A Protein AProtein A

EF-Ts

2Protein A

Hsp70PBP

EF-TuProtein F

3EF-Tu

Protein A

V4BpaS3BpaControl D5Bpa

10 -

15 -20 -

30 -

50 -

80 -

220 -

kDa Marker

1

23

1 1

2

Strategy to identify the YebF transporter

Unnatural protein medicinal chemistry

Directed evolution of the archaea LeuRS system

H2N

NH

N

S OO

O

OHH2N

O

OH

HN

O

H2N

O

OH

HN

H2NOH

OH2N

N

OH

O

H2NOH

O

N

NO

OH

H2N

HN

OH

O

N ON

NO2

H2NOH

O

S

O2N

H2N

B

OH

O

OH

OH

Selection with a single reporter plasmid

Positive selection + Unnatural amino acid+ Cm + Uracil

Survivors containing aaRS capable of charging any natural or unnatural aa on the orthogonal tRNA

Negative selection- Unnatural amino acid

+ 5-FU

Cells that incorporate natural amino acids make toxic product from 5-FU and die;

Cells that incorporate unnatural amino acid only survive on 5-FU

Nex

t rou

nd o

f pos

itive

sel

ectio

n

Deletion of the upp and pyrF genes by the recombinase-based gene replacement

Minimal media+ Uracil

+ Uridine

Minimal media+ Uracil

GeneHog upp pyrF

OMP UMPpyrF

de novo synthesis

Bicarbonate

Uridine

Uracil

uppSalvage Pathway

UDP

UTP

CTP

Summary

• A single plasmid system for the high yield expression of proteins containing unnatural amino acids

• Broad applications depending on the physicochemical properties of unnatural amino acids – chemical and photochemical reactions, spectroscopic probes, novel therapeutics etc.

• Ongoing projects

– Identification of the protein acetylation and secretion pathways

– Unnatural protein medicinal chemistry

– Directed evolution of the leucyl-tRNA synthetase

Acknowledgments

• Minoro Aoshima• Lina Bernal-Perez• Pradeep Budhathoki• Aery Lee

• Kiran Butt• Michael Foster• Brett McKnight• Fatima Sahyouni• Diana Tran

• Dr. Laszlo Prokai (UNTHSC)• Dr. Peter Schultz (TSRI)

TCU (Start-up, RCAF, SERC)

Graduate Students

Undergraduate Students

Collaborators

Financial Support