Three-Part Small-scale Screening Platform for the Masses Ronnie Frederick Center for Eukaryotic Structural Genomics Department of Biochemistry University.

Three-Part Small-scale Screening Platform for the MassesRonnie Frederick

Center for Eukaryotic Structural Genomics

Department of Biochemistry

University of Wisconsin—Madison

NIGMS U54 GM074901, JL Markley, PI, GN Phillips, & Brian G. Fox, Co-Directors

Promega/UW 133-GT34, BG Fox, PI

http://www.uwstructuralgenomics.org

CESG Protein Production

• Unified cell-based and cell-free expression pipelines• Expression screening in E. coli and wheat germ extract• Data captured into Sesame at each step

Modular Vector Design

• Essential modules bounded by unique restriction sites

– Promoter, affinity tags, solubility tags, linker combinations

– In vitro TEV proteolysis of target from other tags

– Over 70 vector variants

Small-Scale Expression Testing

• Small-scale expression evaluations (Studier media)– SDS-PAGE in slab gels (~2 days processing and analysis)

– Tested for total expression, fraction soluble and fraction cleaved

– ~1.5 weeks to evaluate, labor intensive, expensive ($450/ 96 targets)

– No reliable assessment of target status after TEV proteolysis

– Predicting large-scale results ~83% accurate (10% false negative and 6% false positive)

96-well plasmid workgroup 96-plate transformation 96-well growth

Expression and Solubility of MBP-Fusion ProteinsExpression, Solubility, and TEV cleavage

Solubility and TEV Cleavage of MBP-Fusion Proteins

MBP 46 kDa

Tev protease

TargetENLYFQ^S

TargetENLYFQ SMBP 46 kDa

Solubility unknown

Expression & Solubility

Hisn(6-8)

Hisn(6-8)

Results from 66 Targets Passed On to Large-Scale and Grown in 5SM

SS suitable in5SM & TB+g

LSPP suitable22

4

2

IPTG+ 5SS unsuitableLSPP suitable

SS suitable in TB+g

LSPP suitable25

SS suitable in 5SM

LSPP suitable2

Positive in Small Scale but Failed in Large Scale

SS unsuitable in both 5SM & TB+g

LSPP suitable

SS unsuitable in both 5SM & TB+g

(IPTG+)LSPP suitable

6

SS unsuitable in 5SM

LSPP unsuitable

Prediction of Large-Scale Results (Analysis of Medical Relevance Target Workgroup, WG1323)

Total LSPP growths = 66Total LSPP failures = 10Unsuitable in LSPP = 6LSPP Suitable (small-scale error) = 4

Small-scale correctly predicted 49 (suitable) + 6(LSPP unsuitable)

(True positive + true negative = success rate) 49+6/66 = 55/66 = 83.33%

False negative rate = 7/66 = 10.6%False Positive rate = 4/66 = 6.1%

Source of Attrition Within Small-Scale

• For a given vector (VP56K), attrition was due to: poor expression, low solubility and failure in TEV protease cleavage

• Solubility of liberated target is not assessed

1803 Galdieria1888 Cyanidioscyzon merolae (C. merolae)2015 Medical Relevance

0%

20%

40%

60%80%

100%

1817 1876 1888 1938 2015

Workgroups

Breakdown of workgroup suitability

Unsuitable - Weak TevCleavage

Unsuitable - WeakSolubility

Unsuitable - WeakExpression

Suitable

Part 1: A New Modular Vector Design

• Essential elements bounded by unique restriction sites– Promoter, solubility tags, affinity tags, linker combinations– Over 70 expression vector variants

• Linker module altered to contain a TVMV-His8-TEV context

• Constitutive expression of TVMV protease from vector

• Altered expression of the lac repressor (LacI)

• In vivo proteolysis of MBP by TVMV protease (self-cleaving VP62K).

• Auto-induction medium

• First pass Se-Met medium in the production pipeline

• Factorial design identified favored compositions that were different from original PASM (Studier’s media)

• Expression performance is strongly linked to the level of lac repressor (LacI) expressed from the backbone

Part 2: Medium Evolution

Reference: “Enhanced Bacterial Protein Expression During Auto-induction Obtained by Alteration of Lac Repressor Dosage and Medium Composition”Paul G. Blommel, Katie J. Becker, Petar Duvnjak, and Brian G. Fox (in press).

Part 3: Simple robotics for the massesAutomated Purification System

• Can the simple Maxwell robotics contribute to our pipeline?

• Couple the designed vector and the improved expression medium with the new robotics

• E. coli pipeline: Auto-induction in vivo cleavage automated Maxwell purification

• Use it to obtain predictive information of protein behavior in purification, NMR and X-ray structure determination

www.promega.com/paguide/images

Automated Analysis System

• Caliper Lab Chip-90, 1 chip and ~4 hours of screening ($225/96 targets)

• Accurate determination of the amounts of purified protein targets

– Preliminary applications

– Maxwell 16 purified targets

– MBP-fusions (Se-labeled targets)

– TEV-treated 15N-labeled targetshttp://www.caliperls.com/downloads/LC90-BR-01Single.pdf

LC90 Analysis of Maxwell Screen

• Various structural genomics targets or controls

– A mix of the good, the bad and the ugly

• Target protein detected for all 24 targets

• In vivo cleavage with GFP as a control worked

Extending Small-scale screening: Human embryonic stem cell proteins

24152.2214NPM2C1

13459.6114tcl1A8

31035.3258C10 Orf 6A5

87639.8786CCNFA4

Mol. wt.ResiduesProtein Coord. • Human embryonic stem cell protein A8 was expressed in 15N auto-induction medium for an overnight culture from a colony pick

• Automated purification with the Maxwell 16 system gave 3 mg from 12 lanes

Multiple-well purification of 15N labeled Human Embryonic Stem protein (A8)

New Path to Structure Screening: Micro and Meso-scale protein purification

• 15N HSQC NMR of His8-GFP expressed in self-cleaving vector pVP62K at 35 ˚C. The NMR time required was 1 h

• Cost is $50 (labeled medium and purification cartridges). 40 minute run (1.5 mg from 8 mL).

• NMR measurements by Dr. Jikui Song

• 15N HSQC NMR of human embryonic stem cell protein A8 expressed in self-cleaving vector pVP62K.

• The NMR time required was 8 h

• Crystals of SeMet-labeled GFP

Meso-Scale Protein Production and Purification for HSQC NMR Analysis

AcknowledgementsProfessor Brian G. Fox (section supervisor)

Vector Design and FlexiVector Cloning

Paul Blommel, Michael Goren, Peter Martin, Kory Seder, Eric Steffan and Russell Wrobel

Factorial Evolution of Auto-Induction Media

Paul Blommel, Katy Becker and Petar Duvnak

CESG Small-scale screening team

Lai Bergeman, Mike Cassidy, Ah-Young Lim and Jung (James) Whan Yoon, Jason Bunge (High school student) & Dr Jikui Song (NMR analysis)

Past members

John Kunert, Megan Riters, Nick Dillion, Rachel Schiesher & Jay Juhjung Chin