JCIMPT Chang Slides 110107 pdf - JCIMPT Home Pagejit.usc.edu/Shared Documents/RMI_Presentations/RMI_Slides_Chang.pdf · - cDNA clone collection and cloning technology available under

JCSG Center for Innovative Membrane Protein Technologies

Cell-Free Expression

• Used in industry for soluble proteins Swartz, J Microbiol Biotechnol, 33 (7), 476-485, 2006

• Becoming popular for membrane proteins Klammt, C, Schwarz, D, Löhr, F, Schneider, B, Dötsch, V, Bernhard, F, Cell-free expression as an emerging technique for the large scale production of integral membrane protein, FEBS J, 273, 18, 2006

• Expression possible in the presence of certain detergents and/or lipids

• Works very well with small membrane proteins (~20kD), including those that oligomerize.

• Progress towards expression of larger membrane proteins



Cell-Free Expression

• CF Protein expression from DNA plasmids and/or even PCR fragments

• CF Reaction Composition– Lysate– Amino acids and nucleotides– Buffer– ATP and secondary energy source– DNA template

• E. coli and wheat germ are popular• Reticulocyte, insect cell, PURE• Several styles of operation



Cell-Free Expression of Membrane Proteins

1. Grow and lyse cells2. Prepare extract (centrifugation)3. Add detergent/lipids, salts, substrates4. Add template5. Incubate (~ 6 hours)

Klammt et al. (2006) FEBS J Vol 273 (18) 4141-4153


JCIMPT Membrane Protein Cell Free Expression

• Our project is a collaboration with Invitrogen, Inc.- Based on one of the most active bacterial based lysate currently on the market - cDNA clone collection and cloning technology available under this collaboration- Relatively high interest to convert this into commerical product

• Several advantages of the CF system over the in vivo approach- No toxicity issues- High level of control (eliminate “biology”) and reproducibility- Selective labeling (Se, 15N, 13C, deuteration)- Relatively high yields possible (0.6-1.2 mg purified protein per ml reaction typical)- Capable of rapid screening and scaling is linear

• Initial focus on bacterial lysate- Bacterial based lysate is relatively cheap and quick to produce- System takes advantage of bacterial transcriptional/translation efficiency- Introduction of mammalian lipids and also insertion machinery in vitro- Possibility of incorporating membrane protein directly into micelle or other surfactants- No post-translation modifications that introduce heterogeneity- Several sources of lysate are possible and to be developed


• Increased yield of certain membrane proteins (< 20 kD molecular weight)(some up to ~1.5 mg/ml of lysate) (Tier 1)

• Successful to overexpress a much larger range of bacterial membrane proteins of ~40-50 kD molecular weight range. (Tier 2)

• We have increased the efficiency and robustness of our CF system toaccomodate the expression of certain mammalian membrane proteins(~50 kD molecular weight). (Tier 3)

• Tested function of these membrane proteins by ligand binding/microcalorimetry

• Investigated possible alternative lysate sources and the incorporation ofadditional lipids/membranes such as those derived from thykoloid or fromother sources.

• Working with Invitrogen to commercialize for membrane proteins

JCIMPT Cell-Free Membrane Protein Expression Where are we now?



YiiP(33 kD)

EmrD(42 kD)

MscL(14 kD)

EmrE(11 kD)

(1-1.2µg/µl/ml)

(0.6-0.8µg/µl/ml)

Cell Free Expression of Bacterial Membrane Proteins

(43 kD)(40 kD)


EmrE - Small Multidrug Transporter

• First test of our CF system for membrane protein x-ray structure determination

• Bacterial, Small Multidrug Resistance (SMR)• 110 Amino Acid, ~11 kD wild-type• 4 transmembrane helices• Functional as dimer• Critical and conserved residue Glu14 required

for substrate binding/transport• Hetero-dimers like EbrAB are well studied• Controversy on the packing of the dimer



EmrE SeMet Labeling

Wild-type 13986 DLabeled 14181 D

Sulfur 32.06 DSelenium 78.96 D

4 Met labeled with Se

MNPYIYLGGAILAEVIGTTLMKFSEGFTRLWPSVGTIICYCASFWLLAQTLAYIPTGIAYAIWSGVGIVLISLLSWGFFGQRLDLPAIIGMMLICAGVLIINLLSRSTPH 91 and 92

21


CF and in vivo EmrEfunctionally similar

Tryptophan Fluorescence quenching Fluorescence anisotropy Competitive Bindingusing Fluorescence anisotropy


EmrE – New SeMet Data


EmrE - Small Drug Transporter

C2 Crystal Form P21 Crystal Form


EmrE - Small Drug Transporter



EmrE x-ray structure with EM densityUbarretxena-Belandia et al, (2003)

EMBO J 22:6175-6181.


CF Expression of Larger (~50 kD) Membrane Proteins

• Expressed as full-length protein• Stays solubilized if expressed with

– Brij series (35, 58, 78)– Digitonin– Liposomes

• Detergent exchange possible. Some targets may aggregate.

• Proved to be functional after reconstituted into liposome



Human/mammalian targets for cell-free expression

Hybrid transporter/channel. Only known high affinity Cu uptake system in mammals. Anti-cancer drug (cisplatin) transporter. Trimer.

321CTR1 - Copper transport

Solute carrier family 2. Responsible for insulin-regulated glucose disposal.

1255GLUT4 - insulin-regulated glucose transporter

One of a few lysosomal membrane proteins. Transports nucleosides, drugs, steroids, etc. Possible target for development of novel chemotherapeutic agents.

428LAPTM4A - Lysosomal-associated protein transmembrane 4 alpha

Controls organ formation (in the heart) and electrical synchronization (in the brain). Hexamer.

426/32/43CX26/32/43 - Connexin(Gap junction) protein family

SignificanceNo. of TMMW (KDa)Protein



In vitro expression and purification of human membrane proteins using E. coli extract

• Proteins were extracted with Fos-Choline 14 and purified using Ni-NTA column• Yield: 0.5 – 1 mg purified protein/mL of cell-free reaction.


Large-scale expression and purification of GLUT4 using CF without detergent (Mode A)

A. GF peak #1B. GF peak #2

Monodispersed population of CF-expressed GLUT4

Gel filtration profile of Ni-NTA purified GLUT4in Fos-14

Void

Void

M: MarkerP: PelletS: Supernatant

A B


Large-scale CF expression, purification and detergent exchangeof GLUT4 with Brij-35 as solubilization agent (Mode B)

1. Gel filtration peak of GLUT4/Brij-35

Purification of GLUT4 using Brij-35 Detergent exchange of Brij-35-purified GLUT4

1. OG2. UDM3. DDM*4. Cymal 55. Cymal 66. CHAPS

Void

Superdex 200 16/60


0

20

40

60

80

100

120

140

160

180

0 2 4 6 8 10 12 14 16 18 20

3H-SubstrateUV280Conduct

Cell-Free Expression of Mammalian Receptors

ETAROGR1Void

Monomer

Superdex 200 16/60OGR1

Superdex 200 10/30ETAR

~0.2-0.3 mg/ml reaction


Biology and Relevanceof MsbA and Pgp

• ~128kD homodimer

• Shown to bind and hydrolyze ATP

• Transports lipopolysaccharide (LPS), an integral part of the outer bacterial membrane, from the inner leaflet to the outer leaflet of the inner membrane

• MsbA is the only essential ABC transporter identified in gram negative bacteria

• MsbA is a structural and functional homologue of Human P-glycoprotein (Pgp; hMDR1), a clinically important transporter

• Pgp is a multidrug transporter responsible for chemotherapy resistance


NH3

-

+

CO2

TMD

ABC


MsbA Structural Data• Density at 3.7Å• AMPPNP Density at 3.7Å• Hg and Vanadate at 4.5 Å


X-ray Structures of MsbA

Inward Facing Inward Facing Outward FacingAMPPNP and ADP-Vi


Conformational flexibility of MsbA based on structures


Acknowledgements

Srinivas ChittaboinaRitu RoyQinghai Zhang

InvitrogenToni KudlickiJulia Fletcher

Yen-Ju ChenSamantha LieuOwen PornillosAndy ChenAlex Ma

Tuan NguyenAndrey Kariakin

Paul SzewczykXiao He

Andrew WardChristopher L. ReyesJodie YuChristopher Roth

Steve Aller

SSRLALSAPS

NIHNASADOD

Stevens LabYeager LabKuhn LabFinn Lab

Ina UrbatschLab (TTUHSC)

Department of Molecular BiologySkaggs Institute for Chemical BiologyThe Scripps Research Institute