A comparison of multiple E. coli protein expression systems using

orthologous plant proteins

Belinda Sharpe

Division of Biology

Faculty of Natural Sciences

Imperial College London

EDS1

• Functions as part of a signalling pathway in plant defence responses

• Part of a family of related proteins, including PAD4 and SAG101

• All share a similar domain structure from sequence homology

• EDS1 can physically interact with PAD4, SAG101, and also with itself

lipase-like domain EP domain

EDS1

• Initially focussed on full-length EDS1, aiming towards crystallisation trials

• Full length AtEDS1 with N-terminal His-tag (pET15b)

• High levels of expression (~30 mg/L), expressed in both soluble and insoluble fractions

• Purification of soluble protein was successful, and appeared monomeric by gel filtration

• Was not stable over time and no success in crystallisation trials

EDS1

EDS1-SAG101 complex

• Given EDS1 was unstable in isolation, attempt co-expression of EDS1 with protein binding partners (PAD4 and SAG101)

• Co-expression and co-purification of EDS1 with His-tagged SAG101 was successful

• However, the complex precipitated in crystallisation trials

EDS1SAG101

97

66

45

kDa

Fragments of EDS1

~40 kDa fragment

~30 kDa fragment

3541

80

623

Full-length EDS1 (73 kDa)

~40 kDa fragment

~30 kDa fragment

• Use limited proteolysis to identify smaller stable fragments of EDS1

• Two stable fragments were identified using combination of N-terminal sequencing, MS, and size estimation by SDS-PAGE

354

Next step…

1. Compare expression and solubility of full length EDS1 and smaller defined EDS1 fragments

2. Use different expression vectors, with different affinity tags

3. Compare Arabidopsis EDS1 to another EDS1 orthologue

1. EDS1 constructs

~40 kDa

~30 kDa

354

1

80

623

1

354

~73 kDaEDS1-Full

EDS1-Long

EDS1-Short

2. New expression vectors

• Gateway system

• Based on in vitro site-specific recombination system

• Allows you to move your gene of interest into one “entry” vector, then easily into multiple vector systems for functional analysis and protein expression

2. New expression vectors

GST EDS1 construct

A TEV protease site was engineered into the sequence, to allow for cleavage of each tag if required

EDS1 construct6xHis

MBP EDS1 construct6xHis

3. Choosing an orthologue

• EDS1 is found in Arabidopsis, tomato, tobacco, barley, wheat, rice, and others.

• EDS1 in tomato has been shown to have the same function as EDS1 in Arabidopsis.

• Sequences are 60% similar, 42% identical.

Summary of constructs

Arabidopsis

partly soluble

Tomato

6xHis GST MBP6xHis

EDS1S

EDS1L

EDS1F

EDS1S

EDS1L

EDS1F

Induction conditions

• Various induction conditions trialled IPTG concentration (50 μM – 1 mM)

Made no significant difference to levels of expression

Temperature (20 and 37°C)Constructs were completely insoluble at 37°C, however some soluble protein was observed at 20°C

Comparison of EDS1 constructs

• Although His-tagged full-length EDS1 was partly soluble, His-tagged smaller domains of EDS1 were not.

EDS1S EDS1L EDS1F

sol ins

6xHis

97

66

45

30

kDa

insoluble insoluble partly soluble

Comparison of vectors

6xHis GST MBP6xHis

• Soluble protein for HisMBP construct only

97

66

45

30

kDa

insoluble insoluble partly soluble

EDS1L

Comparison of orthologues

• Differences between soluble protein expression for Arabidopsis and tomato constructs were seen for the HisMBP-EDS1L fusion protein

MBP EDS1L6xHis

insolublepartly soluble

Summary of results

Arabidopsis insoluble insoluble partly soluble

insoluble insoluble partly soluble

partly soluble

Tomato

insoluble insoluble insoluble

insoluble insoluble partly soluble

6xHis GST MBP6xHis

EDS1S

EDS1L

EDS1F

EDS1S

EDS1L

EDS1F

Summary

• Did see difference in expression of soluble protein between different constructs, vectors, and between Arabidopsis and tomato

• Given initial results for full-length EDS1, there was no dramatic shift in the percentage of soluble and insoluble protein expressed

• Next… look at other tags (pET32-Trx), and explore expression in a different system to E.coli

Thanks to…

Bart Feys

Nick Brereton

Vincenzo Salerno