Lessons from the Quest for Quality Protein(and Crystals)

Pure

Homogeneous & Stable

Monomer

Dimer

8mM excess OG micelles

PDC280nm

SDX, 7.3, 40mM OG

RI

Minimized [Detergent]

Well behaved

IE

Low [OG]/no phase sep

SEC microinjections

280nm

DiluteConcentrated

Over time

High [OG]/phase sep

PerspectiveThe Challenge

Detergents/lipids complicated & barely understoodPDC not homogenousProtein, Detergent belt, Micelle and Crystal packing dependant on many parameters

Primary and secondary detergent/lipid (type & conc.)Ionic strength (type and conc.)Osmolytes, additives, precipitating agentsTemperature, protein

Burov et al 2008

C8TMA-Cl

C16TMA-Cl

Sansom et al. 2005

KvAP/DM Simulation250 ns snapshot

OG phase diagram (Zulauf 1991)

PEG

Pebay-Peyroula et al 1995

Crystal packing dependant on detergent

C10DMA0

OG

Sauer et al, Acta Crystal D, 2002

C8E12/C8-2-HES

OmpF

Working FoundationDo whatever it takes to obtain/maintain PHS with minimized detergent

May take 2-4 steps which can remove “all” endogenous lipids Not concerned with initial lipid removal

+/- lipid will not inhibit xtal growth, but xtal qualityIt’s requirement will be determined thought the purification processSecondary detergents/lipids can be added back during crystal trial

EmpiricalNo one/few magical condition amendable to all targetsEvery protein needs to be considered independently

Map out solubility/crystallization space using different crystallization methods VD, batch, dialysis, LPC, counter diffusion

If quality protein but poor or no crystalsSystematically modify the PDC

1st : detergent belt2nd: protein

Experimental styleChromatographyQuality Output (careful, complete and methodical; slow)

Go-fast, streamlined med-high throughput very important

Purification & CharacterizationDetergent Solubilization

Concentration

Membrane Preparation

250mM OG -- 40mM OG solvent20mM DDM -----------------------20mM C14PC/C12PC/MMPC--

Ni, AffinityDesalt

Tag CleavageCleanup

Size Exclusion

Desalt/pH change

Cation and Anion Exchange

Tetra Detector Array/Analysis

Concentration: Abs & RI

Shape (IV), size (Rh): Viscometer

Mass: RALS

Size Exclusion

Key Parameters Detergent/lipidpHIonic strengthReducing agentOsmolytesAdditives

Max MWt cut-off filter

IE, Ni, Affinity

Dialysis

Crystallization & Crystallography(CSMP)

Core Purification ApproachProperly targeted Over expressed ≥ 500ml culture

All fractions 9-15runs /exp/3day

(Minimal 3 Detergent Screen)

Concentrate100kDa start

0.5-2mM DDM solvent

Start10% glycerol

5mM BME/2mM DTT if Cys present

PurificationpH/salt solubility/homogeneityDetergent exchangeWell behaved

Wash, LyseOptional buffer & high salt washMembrane Signature Gel

+TLCand/or

+Dilution Factor

With Corey Anderson, André Bachmann, Sotiri Banakos, Akanksha Bapna, Sarika Chaudhary, Melissa Del Rosario, Vladimir Denic, Robert Edwards, Pascal Egea, Franz Gruswitz, Frank Hays, Joe Ho, David Julius, Monty Krieger, Witek Kwiatkowski, John Lee, Min Li, Bipasha Mukherjee, Vinod Nair, Zach Newby, Roger Nicoll, Sabrina Noel, Joseph O’Connell, Yaneth Robles, Edwin Rodriquez , Zygy Roe-Zurz, Renee Robbins, David Savage, Shimon Schuldiner, Tomomi Tsomeya, Linda Vuong, Jonathan Weismann, and Ronald Yeh.

People with italicized names are no longer working with us.

High Priority MPEC Protein Progress

Structure

Diffraction

Crystal

PHS

SE, IE

Tag Cleave

Affinity

Solubilize

Expression

S. cere, HEK, P. past, E. coli, Homologs /E.coli

MPEC Targets (>128, 32 PHS)

2.1 1.8 2.0 Å

3.8 3.5 10

S. cere HEK P. past E. coli Homologs /E.coli

Wo

rkfl

ow

Membrane Preparation

Purification ApproachProperly targeted Over expressed ≥ 500ml culture

Wash, LyseOptional buffer & high salt washMembrane Signature Gel

≥ 500ml culture (scale up issues)

Membrane signature gel (high to low conc.)

Wash membranes as much as neededLow and high salt washes

Rachel Bond

Detergent Solubilization

250mM OG -- 40mM OG solvent20mM DDM -----------------------20mM C14PC/C12PC/MMPC--

Purification ApproachStart

10% glycerol 5mM BME/2mM DTT if Cys present

Only small subset of detergents initially required7 for full gel

OG, DDM, FC12, MMPC, CHAPSO, C12E8, LDAO

Keep in mind theLarge available arsenal for solubilization & purification

(Thank you Anatrace, Qinghai Zhang, Sam Gellman)

Common purification solvents40mM OG, 18mM NG, 8mM DM,0.5-2mM DDM, 2-4mM FC12, 0.5mM FC14exploring MMPC, MMPG, mixtures

Purification & Characterization

Size Exclusion

Desalt/pH change

Cation and Anion Exchange

Key Parameters Detergent/lipidpHIonic strengthReducing agentOsmolytesAdditives

Purification Approach

All fractions 9-15runs /exp/3day

Concentrate100kDa start

Start10% glycerol

5mM BME/2mM DTT if Cys present

PurificationpH/salt solubility/homogeneityDetergent exchangeWell behaved

Concentration (find max kDa)100 to 50 to 30 kDa spingel, spec

Remove heterologous residuesAlways test different pHs

Minimally desalt into other pHs

Ni, AffinityDesalt

Tag CleavageCleanup

Ni bump desalted to pH 6, 8, and 9

Post TEV cleavage at pH 6, 8, and 9

First pass of human/DDM (Rachel Bond)

pH 5

pH 7

pH 9

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

-5 5 15 25 35 45 55 65 75 85 95

Time (min)

OD 280nm (2.5mm path)

Always ask if SEC peaks run true

Stable GlpF/OG; 2.2 Å

Post Ni, no desalt

Fraction 4

Day 1 Day 2

SEC/OG

SEC/DDMFraction 3+5

Human/DDM Rebecca Robbins1st purification pass

F4

Concentration

Tetra Detector Array/Analysis

Concentration: Abs & RI

Shape (IV), size (Rh): Viscometer

Mass: RALS

Size Exclusion

Max MWt cut-off filter

IE, Ni, Affinity

Dialysis

Crystallization & Crystallography

Purification Approach

Always follow SEC profile during protein concentrationDoes purified, homogenous protein remain stable upon concentration?

Good

Bad

Concentration

Tetra Detector Array/Analysis

Concentration: Abs & RI

Shape (IV), size (Rh): Viscometer

Mass: RALS

Size Exclusion

Max MWt cut-off filter

IE, Ni, Affinity

Dialysis

Crystallization & Crystallography

Purification Approach

“Universal Calibration Method”Vh=IV*M vs. retention time

Still some exemptions SE matrix not inert

Chromatography can be successfully to concentrate while minimizing [detergent]

Poster/ manuscript/website: 4 proteins, 3 detergents, 4 different methods

RI detectors ROCK! Every workstation should have one!

Quantitate excess [detergent] while following PDC homogeneity

PDC systems must be used when studying micelle behavior on MWCO filters

+TLCand/or

+Dilution Factor

Sold on the concept of multi detection for SEC

PDC mass, size, shape and % binding partners using SEC

Micelle mass, size, shape

But Tetra detection not ready for mainstream MP work

Acquisition fine

Slow and involved

No fraction collection (when analyzing)

Analysis OK, but complicated

Requires calibration standard

Very sensitive (sees everything)

Accuracy problematic (assumptions must be verified)

Calibration Standard: dn/dc, dA/dc, Mass

Unknown MP: dA/dc, dn/dc

Totally willing to keep moving forward with tetra detection

Ovalbumin MWt (44,300 Da actual)

Mw (weighted avg)44183 whole peak analysis34392 peak slice analysis

For PDC analysis, obtain single UV peak before proceeding with Multi detectionPeak slice analysis can only be used when Mass constant throughout peak

humanMP/OG

191,012 PDC/110,792 protein (3.2 monomers/PDCwhole peak analysis

179,652 PDC/113540 protein (3.3 monomers/PDC) peak slice analysis

Mw/Mn of single peaks = 1.009 = monodisperse

MWt distribution Mw/Mn = 1.001 = monodisperse

Acknowledgments

MPEC Subproject 6: Protein Purification

Bill Harries & John Lee (infrastructure, management)Pat Greene (consulting, grants)

Olivia Viloria (finance)Suzan Betheil (admin)

and Robert Stroud (Commander & Chief)

Andrew Sandstrom(University of Chicago)

Collaborators

Rebecca Robbins, Mimi Ho, Rachel Bond