P4EU Heidelberg 2016, June On-column protein cleavage: The Profinity and bdsumo systems. Tamar Unger.

Transcription

1 P4EU Heidelberg 2016, June On-column protein cleavage: The Profinity and bdsumo systems Tamar Unger

2 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

3 Tag and Fusion Proteins small tags and/or big fusions detection and/or purification 6His 0.8KDa Ni column GFP 23kDa GST 26kDa Glutathione Trx 11kDa CBP 20kDa Cellulose Chitin 5.6kDa Chitin SUMO 12 kda Profinity 8 kda MBP 40kDa amylose Halo 34kDa NusA 54.8kDa Other

4 Protease recognition cleavage sites Enterokinase D-D-D-D-K-X/ Thrombin Factor X TEV X-X-P-R/K-X-X I-E/D-G-R-X E-N-L-Y-F-Q-/G Profinity E-E-D-K-L-F-K-A-L/ bdsenp1 bdsumo

5 Profinity exact- expression/purification system (Bio-Rad) Ruan B. et al Biochemistry, 2004, 43 (46), pp 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. Potential: A tag-free protein in a single purification process

6 The Profinity system Tag His-tag Profinity-tag MCS ppal7/ppal8 pet28-profinity Amp R Kan R petgst-profinity Kan R pettrx-profinity Kan R petgb1-profinity Kan R Construction of multiple expression vectors for improved expression Peleg et al. 2016, Methods in Molecular Biology

7 The Profinity system kda M S E S E S E S E Profinity pet28 pettrx petgst petgb1 vector Peleg et al. 2016, Methods in Molecular Biology

8 Remarks for the Profinity exact system 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 H 3 PO 4 following re-equilibration with binding buffer containing 100 mm sodium phosphate buffer.

9 Advantageous Advantageous and disadvantageous One step purification Native N-terminus of the recombinant protein Disadvantageous Partial cleavage, obtaining the digested as well as the fused protein Non-ideal N-terminal amino acids of protein may affect cleavage

10 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 25 o C for up to 3hr or ON at 4 o C Steffen Frey and Dirk Gorlich, J. Chromatography (2014), 1337:95-105

11 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

12 Procedure Capture His-bdSumo-TOI on Ni-column Wash contaminants Elute fusion protein Cleave on-column by adding bdsenp1 protease w/o His Cleave in solution by adding His-Tev-bdSENP1 protease Capture on Ni-column the fusion and protease Collect the unbound material which contains the native TOI Collect the unbound material which contains the native TOI

13 bdsumo-toi Ni-purification Dialysis bdsumo cleavage Incubation of 1hr at 25 o C Incubation of 3hr at 25 o C fusion fusion target Sumo target Sumo tag tag

14 On-column purification of Sumo-TOI bdsumo-toi fusion cleaved

15 bdsumo-toi fusion cleaved

16 bdsumo fusions TOI1 TOI2 TOI3 TOI4 TOI5 TOI1 pet28-toi2

17 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

18 A novel computational strategy for protein stability in E. coli Automated structure and sequence-based design of proteins for high bacterial expression and stability by Adi Goldenzweig and Sarel Fleishman 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

19 DNA manipulations by the RF methodology Unger T et al., (2010) J Struct Biol 172:34 44

20