P4EU Heidelberg 2016, June 15-16

On-column protein cleavage: The Profinity and bdSumo systems

Tamar Unger www.weizmann.ac.il/ISPC

Outline of talk:

Description of the Profinity System; Principles, vectors, procedure and examples

Description of the bdSumo System; Principles, vectors procedures and examples

A new algorithm for designed protein stability

DNA manipulation by the RF methodology

Tag and Fusion Proteins

• small tags and/or big fusions

• detection and/or purification

• 6His 0.8KDa Ni column

• GST 26kDa Glutathione • Trx 11kDa • SUMO 12 kDa • CBP 20kDa Cellulose

• NusA 54.8kDa

• GFP 23kDa

• Chitin 5.6kDa Chitin • Profinity 8 kDa

• MBP 40kDa amylose • Halo 34kDa

• Other

Protease recognition cleavage sites

• Enterokinase D-D-D-D-K-X/

• Thrombin X-X-P-R/K-X-X

• Factor X I-E/D-G-R-X

• TEV E-N-L-Y-F-Q-/G

• bdSENP1 bdSumo

• Profinity E-E-D-K-L-F-K-A-L/

Profinity eXact- expression/purification system (Bio-Rad)

Potential: A tag-free protein in a single purification process

Ruan B. et al Biochemistry, 2004, 43 (46), pp 14539–14546

The system is based on the immobilized Subtilisin engineered mutant (S189) protease, which recognizes and binds the engineered affinity tag prodomain of Subtilisin fused to the N-terminus of the target protein. Thus the Subtilisin is used as both the affinity ligand and the processing protease. The process includes: I. Cloning the engineered 8.2 kDa Subtilisin prodomain at the

N-terminus of the target of interest, and expression in bacteria.

II. Binding of the fusion protein to the immobilized Subtilisin column.

III. Washing to remove free contaminants from the column. IV. Cleavage on the column, precisely at the C-terminus of a nine

amino acid sequence (EEDKLFKAL) corresponding to the Subtilisin prodomain, in the presence of fluoride anions to release the target protein.

The Profinity system

His-tag Tag Profinity-tag MCS

pPAL7/pPAL8 AmpR

pET28-Profinity KanR

pETGST-Profinity KanR

KanR pETTrx-Profinity

KanR pETGB1-Profinity

Construction of multiple expression vectors for improved expression

Peleg et al. 2016, Methods in Molecular Biology

The Profinity system

Peleg et al. 2016, Methods in Molecular Biology

M S E S E S E S E

Profinity pET28 pETTrx pETGST pETGB1

vector

kDa 180

130

100

70

55

40

35

25

15

10

Additional linker (Thr-Ser) following the Profinity-tag might be needed to improve cleavage.

Buffer for cell lysis should NOT contain NaCl or KCl or Tris-Cl which trigger the cleavage process. We use 100 mM Sodium phosphate buffer pH-7.2 for binding/washing.

Cleavage can also be performed by 10 mM sodium azide.

Column regeneration- washing with 0.1 M H3PO4 following re-equilibration with binding buffer containing 100 mM sodium phosphate buffer.

Remarks for the Profinity eXact system

Advantageous and disadvantageous

• One step purification

• Native N-terminus of the recombinant protein

Advantageous

Disadvantageous

• Partial cleavage, obtaining the digested as well as the fused

protein

• Non-ideal N-terminal amino acids of protein may affect cleavage

His-Sumo protein fusion

14x His Sumo Protein

Sumo protease (w/o His tag)

The protease is highly efficient: 1:1000 ratio is used Cleavage at 25oC for up to 3hr or ON at 4oC

Steffen Frey and Dirk Gorlich, J. Chromatography (2014), 1337:95-105

The Brachypodium distachyon bdSUMO/bdSENP1 system

His14-bd-Sumo fusion 14 kDa

His-Tev-bdSENP1 protease 32 kDa (without His-Tev 28 kDa)

Recommended cleavage buffer: 250 mM NaCl, 40 mM Tris/HCl pH7.5, 2 mM MgCl2, 250 mM sucrose, 2 mM DTT

Enzyme is very efficient at least 1:10000 enz:substrate

Broad range of buffers and temperatures

Procedure

Capture His-bdSumo-TOI on Ni-column

Wash contaminants

Cleave on-column by adding bdSENP1 protease w/o His

Collect the unbound material which contains the native TOI

Elute fusion protein

Cleave in solution by adding His-Tev-bdSENP1 protease

Collect the unbound material which contains the native TOI

Capture on Ni-column the fusion and protease

Incubation of 1hr at 25oC

bdSumo-TOI • Ni-purification • Dialysis • bdSumo cleavage

fusion

target

Sumo

tag

Incubation of 3hr at 25oC

fusion

target

Sumo

tag

fusion

cleaved

On-column purification of Sumo-TOI

bdSumo-TOI

cleaved

fusion

bdSumo-TOI

TOI2 pET28-TOI2 TOI1 TOI3 TOI1 TOI4 TOI5

bdSumo fusions

Conclusions

On-column cleavage makes purification more time-efficient

The Profinity system requires purchasing of specific columns from Bio-rad

The bdSumo system facilitates the purification of recombinant proteins in E.coli

10- to 1000-fold higher specific activity than TEV protease

Efficient tag removal also at 0 °C and in high-salt buffers

Production of target proteins with any N-terminal residue including Proline

Automated structure and sequence-based design of proteins for high bacterial expression and stability by Adi Goldenzweig and Sarel Fleishman

A novel computational strategy for protein stability in E. coli http://pross.weizmann.ac.i

Designed hAChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ~2,000-fold higher levels in E. coli compared to wthAChE. The dAChE exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography

DNA manipulations by the RF methodology

Unger T et al., (2010) J Struct Biol 172:34–44