Powerful SUMO Protein Expression Systems

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1 Powerful SUMO Protein Expression Systems Introduction: Presentation: Ali el Baya, Ph.D. Sales Manager Protein related Services & Reagents tebu-bio Mark Bell, PhD Business Development Manager LifeSensors, Inc.

2 Before we begin User console Audience audio is muted Post questions to our staff anytime via Chat Slides and recorded session available soon

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4 SUMO Fusion Tag Platform for Enhancing Recombinant Protein Expression What is SUMO? How does it work? Our products Examples of use

5 SUMO Fusion Tag Platform for Enhancing Recombinant Protein Expression

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7 What is SUMO? SUMO (Small Ubiquitin-like MOdifier) is a member of the ubiquitin family: SUMO (smt3) of yeast husumo1, husumo2/3, and husumo4 NEDD8, ISG15, etc. Like ubiquitin, SUMO is attached to target proteins post-translationally through an isopeptide bond Unlike ubiquitin, SUMO is not involved in targeting proteins for degradation SUMO is involved in regulating diverse protein functions, including solubility and chaperone functions

8 SUMO Protease Intracellular cysteine protease Involved in: - Maturation of SUMO precursor - Deconjugation of SUMO Protease recognizes tertiary structure of SUMO Cleaves after a Gly-Gly sequence at the C-terminus of SUMO, resulting in highly specific processing Protease cleaves with high efficiency (high K cat ) SUMO and SUMO proteases are found in all eukaryotes

9 What is SUMOpro? Original SUMO system Smt3 from yeast Only works in prokaryotic expression systems SUMOpro is cleaved by SUMO Protease 1 (Ulp1) All of the benefits SUMO is known for: o Increased soluble expression yield o Efficient tag removal with no amino acid residues o Simple purification stream

10 What is SUMOstar? Extends all of the SUMO benefits to eukaryotic systems Eukaryotes have endogenous SUMO proteases SUMO is highly conserved; processed in all systems SUMOstar mutated and not recognized by native SUMO proteases SUMOstar protease carries mutations that compensate for the mutations in SUMOstar

11 SUMOpro versus SUMOstar SUMOpro is cleaved by SUMO Protease 1 o Works in prokaryotic systems like E. coli SUMOstar is cleaved by SUMOstar Protease 1 o Works in all systems, including E. coli, yeast, insect, and mammalian cell types

12 SUMO process overview

13 SUMOstar cleavage by SUMOstar protease SUMOstar protease U P kd SUMOstar-GFP SUMOstar protease GFP SUMOstar 14 6

14 Commonly use tag removal enzymes Protease Cleavage site Location Residual AAs TEV, AcTEV, ProTEV Thrombin (fiia) Factor Xa (fxa) Enterokinase (EK) SUMO proteases ExxYxQ/(G/S) LVPRG IEGR/x DDDDK/x C-terminus of SUMO/x N C N C N C N C G/S ExxYxQ G LVPR none IEGR none DDDDK ph range/ optimum Enz:target $/mg POI /7 1-3% (wt/wt) % (wt/wt) % (wt/wt) % (wt/wt) N only none % (wt/wt) 3.15 TagZyme XaaYaaGln N only none enz Immobilized Subtilisin BPN C-terminus of propeptide/x N only none 7.2 1:1 (mol/mol) 26.00

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16 Our Products

17 E. Coli o SUMOpro or SUMOstar Our Products by system o Either Amp or Kan resistance Yeast o Intracellular SUMOstar (Saccharomyces) o Secretory SUMOstar (Pichia) Insect / BEVS o Both Intracellular and Secretory SUMOstar Mammalian o Both Intracellular and Secretory SUMOstar

18 Our Products by type Individual items o Vectors o Proteases (250, 500, 1000, 5000 unit sizes) o Anti-SUMOpro/SUMOstar antibody Kits (everything you need to get started!) o Vector of choice o 250 units of corresponding protease o Anti-SUMOpro/SUMOstar antibody o Control protein (SUMOpro or SUMOstar fused to GFP

19 Examples of Use

20 Over 200 examples of proteins expressed with SUMO

21 Specific examples of proteins showing enhanced expression with SUMO Peptides Bosse-Doenecke, et al. Prot. Exp. Purif., : Brazzein, sweet protein from corn Assadi-Porter, et al. Prot. Exp. Purif., : Cytokines Kirkpatrick, et al., Prot. Exp. Purif., (1): Sun, et al., Appl Microbiol Biotechnol, : Chemokines Lu,et al., Prot. Exp. Purif., : Viral proteins Assenberg, et al., Acta Cryst., F64: Single chain antibody Ye, et al., Appl Microbiol Biotechnol, :

22 SUMO fusion enhances expression and solubility Marblestone et al., Protein Sci. 2006

23 Expression of contract protein in E. coli Lysate IEX1 HT1 IEX2 Digest Prod. Prod. - βme kda Improved expression of Protein of interest (POI) from 8% TCP to >20% TCP Purification reduced from 5 column steps to 4 Overall yield improved from ~16% to >40% SUMO-POI POI

24 SUMOstar enhances expression and solubility in yeast kd 100 S. cerevisiae P. pastoris MW H H S S S* S* kd 100 MW S* S* H H SUMO* -GFP SUMO*- GzmB 30 GFP 30 GzmB 20 GFP Blot 20 GzmB Blot Intracellular Secreted H His 6 S His 6 -SUMO S* His 6 -SUMOstar

25 SUMOstar enhances expression in Sf9 cells kd GFP H S S* kd UBP43 H S S* SUMOstar- GFP SUMOstar- UBP43 UBP43 31 GFP Intracellular Intracellular H His 6 S His 6 -SUMO S* His 6 -SUMOstar

26 Peroutka et al., Protein Sci 2008 Expression of mpla 2 Group X in HEK293

27 Accelerated production of subunit vaccine antigens using SUMO DARPA/DTRA contract for Accelerated Manufacture of Pharmaceuticals

28 Accelerated production of subunit vaccine antigens using SUMO Anthrax Recombinant Protective Antigen (rpa)

29 Accelerated production of subunit vaccine antigens using SUMO

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31 Open questions

32 You will recieve the SUMO presentation shortly Vísit tebu-bio s SUMO Tech page: Benefit from our special offer for participants: 30% off until 15/06/2013! For any request contact you tebu-bio office in Europe: Belgium France Germany Italy Netherlands Nordic countries Portugal Spain UK & Ireland From other European countries contact: europe@tebu-bio.com

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