The novel Fh8 tag: a novel fusion system for recombinant protein production and purification in Escherichia coli

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1 The novel Fh8 tag: a novel fusion system for recombinant protein production and purification in Escherichia coli Sofia J. Costa th P4EU meeting

2 Fh8 and H tags Fh8: 8-kDa protein from Fasciola hepatica Excreted-secreted antigen by the parasite F. hepatica in the early stages of infection Located on the surface of the parasite useful tool for the diagnosis, vaccine and drug development against F. hepatica infections Immunogen strong immune response Belongs to the calmodulin-like EF-hand Calcium Binding Protein family Sofia J. Costa 2

3 Fh8 and H tags Fh8: 8-kDa protein from Fasciola hepatica * ** Local 1 - Ca 2+ + Ca 2+ EF-1 EF-2 Local 2 * Zhou, Y., Frey, T.K. and Yang, J.J. (2009) Viral calciomics: interplays between Ca 2+ and virus. Cell Calcium 46, 1-17 ** Fraga, H., Faria, T.Q., Pinto, F., Almeida, A., Brito, R.M.M. and Damas, A.M. (2010) FH8-a small EF-hand protein from Fasciola hepatica. Febs Journal 277, M S M P S S P S V V Q E V E K LL H V L D R N G D G K V S A E E L K A F A D D S K C P L D S N K I K A F I K E H D K N K D G K L D L K E L V S I L S S H Hypotheses: - Important for Fh8 s stability and production; - Critical for the immunological response of the Fh8 antigen. Sofia J. Costa 3

4 Fh8 and H tags Protein expression in E. coli Protein purification Antibody production Initially: Fh8 and H tags protein expression in E. coli H-fused proteins elicited a humoral response: production of polyclonal antibodies Sofia J. Costa 4

5 Fh8 and H tags Protein expression Solubility + in E. coli Protein purification More recently: Fh8 and H peptides as solubility enhancer partners, and comparison to the traditionally used solubility tags; The Fh8 usefulness as a purification handle. Sofia J. Costa 5

6 Comparison with traditional fusion tags Costa, S. J., et al. (2013). Applied Microbiology and Biotechnology 97(15): CP12-1 Surface protein from Cryptosporidium parvum parasite His 6 GST MBP SUMO NusA Trx Fh8 H FTL - 1 Lectin from Artocarpus incisa plant Sofia J. Costa YPK1, YPK2, SPO14, RVS167-4 proteins from Saccharomyces cerevisiae (membrane, cytoplasm, and nucleus) 6

7 Comparison with traditional fusion tags Costa, S. J., et al. (2013). Applied Microbiology and Biotechnology 97(15): T7 promoter Lac operator Fh8 or H RBS ATG 6xHis Fh8 TEV MCS 6xHis petm Fh8: 10 kda petm H: 3.6 kda NcoI-KpnI-BamHI-EcoRI-SacI-NotI-XhoI 8 tags ( kda) X 6 target proteins ( kda) 10 and 500 ml cultures 4 E. coli strains and 2 induction conditions Purification by nickel affinity Tag removal using the TEV protease (or SenP2 protease for SUMO fusions) Sofia J. Costa 7

8 Soluble Expressão Expression solúvel Expressão Soluble Expression solúvel - pontuações scores e 3 Comparison with traditional fusion tags Small scale: Costa, S. J., et al. (2013). Applied Microbiology and Biotechnology 97(15): SDS-PAGE Bradford 1 1 Scale 0-3 0,9 0,8 0,7 0,6 0,5 0,4 RIL Rosetta Tuner SoluBL21 0,9 0,8 0,7 0,6 0,5 0,4 RIL Rosetta Tuner SoluBL21 0,3 0,3 0,2 0,2 0,1 0,1 0 CP12 FTL YPK1 RVS167 SPO14 YPK2 0 CP12 FTL YPK1 RVS167 SPO14 YPK2 Best induction conditions: 0.2 mm IPTG, 18 ºC, 20 h (except CP12: 0.5 mm, 28 ºC, 3 h) Sofia J. Costa 8

9 Tag Soluble Expression - scores 2+3 Comparison with traditional fusion tags Small scale: Costa, S. J., et al. (2013). Applied Microbiology and Biotechnology 97(15): Expression: MBP ~ NusA ~ Trx > Fh8 ~ H ~ His Solubility: MBP > NusA > Fh8 ~ Trx > GST ~H > His 100% 90% 80% 70% 60% 50% 40% 30% RIL Rosetta Tuner SoluBL21 20% 10% 0% His Trx GST MBP NusA SUMO H Fh8 Sofia J. Costa 9

10 Comparison with traditional fusion tags Large scale: Costa, S. J., et al. (2013). Applied Microbiology and Biotechnology 97(15): FTL His Trx NusA H Fh8 His Trx NusA H Fh8 CP12 Fh8 ~Trx ~NusA 21 kda 32 kda 74 kda 66 kda After tag removal: Fh8 ~ Trx > NusA YPK2 MBP H Fh8 27 kda 23 kda 122 kda 21 kda 18 kda 12 kda 12 kda 88 kda 81 kda YPK1 His NusA Fh8 NusA H Fh8 RVS kda NusA > Fh8 111 kda 57 kda 64 kda 46 kda After 39 kda tag removal: NusA > Fh8 Fh8 > MBP kda After tag removal: Fh8 > MBP Sofia J. Costa 10

11 The Fh8 tag as purification handle Costa, S. J., Coelho, E., et al. (2013). Protein Expression and Purification. doi: /j.pep GFP (green fluorescent protein) and SOD (superoxide dismutase) Hydrophobic interaction vs Nickel affinity Fh8: Without Ca 2+ With Ca M M kda kda sample loaded onto the purification column 2 flow-through sample 3 washing sample 4 elution with EDTA 5 elution with ph 10 Ca 2+ -dependent binding Sofia J. Costa 11

12 Purification Efficiency % Fluorescence (RFU/mg of protein) Purification Efficiency % Fh8GFP and Fh8SOD: 3000 The Fh8 tag as purification handle Costa, S. J., Coelho, E., et al. (2013). Protein Expression and Purification. doi: /j.pep % 90% 80% 70% 60% 50% 40% 30% IMAC HIC 100% 90% 80% 70% 60% 50% 40% 30% IMAC Nickel affinity IMAC HIC Hydrophobic interaction Fh8-HIC HIC/IMAC IMAC HIC 20% 10% % 10% 0% Fh8GFP 0 HisGFP 0% Fh8SOD Fh8GFP HisSOD IMAC/HIC or HIC/IMAC: protein purity Sofia J. Costa 12

13 Fluorescence (RFU/mg of protein) Fluorescence (RFU/mg of protein) SOD Activity (U/mg of protein) Fh8GFP and Fh8SOD: HisGFP Fh8GFP Fh8SOD(control) 0 The Fh8 tag as purification handle Costa, S. J., Coelho, E., et al. (2013). Protein Expression and Purification. doi: /j.pep HisSOD Fh8SOD Fh8GFP(control) Fh8GFP IMAC Nickel affinity IMAC Proteins kept their biological activity after purification by Fh8-HIC. HIC Hydrophobic interaction Fh8-HIC HIC/IMAC Sofia J. Costa 13

14 Conclusions The novel Fh8 tag: incresases protein expression levels in E. coli promotes/improves protein solubility is ranked among the best traditionally used solubility enhancer tags offers an efficient and economical protein purification one of the few existing tags to combine solubility enhancer and purification abilities Sofia J. Costa 14

15 Conclusions The novel Fh8/H fusion system: Expression & Solubility purification immunogenecity into the two lowest molecular weight fusion partners described so far Sofia J. Costa 15

16 Thank you for your attention! about.me/sofiacosta Questions? Sofia J. Costa 16