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M. HAERTLEIN, INSTITUT MAX VON LAUE - PAUL LANGEVIN 3 rd D-Lab Meeting, 28 th April 2003 EMBL Heidelberg Optimisation of auxotrophic strains for expression.

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Presentation on theme: "M. HAERTLEIN, INSTITUT MAX VON LAUE - PAUL LANGEVIN 3 rd D-Lab Meeting, 28 th April 2003 EMBL Heidelberg Optimisation of auxotrophic strains for expression."— Presentation transcript:

1 M. HAERTLEIN, INSTITUT MAX VON LAUE - PAUL LANGEVIN 3 rd D-Lab Meeting, 28 th April 2003 EMBL Heidelberg Optimisation of auxotrophic strains for expression of proteins with specifically deuterated amino acids The production of  C -crystallin with deuterated tryptophan residues Marie-Thérèse Dauvergne (EMBL) Michael Haertlein (ILL) Peter Timmins (ILL)

2 M. HAERTLEIN, INSTITUT MAX VON LAUE - PAUL LANGEVIN 3 rd D-Lab Meeting, 28 th April 2003 EMBL Heidelberg DE3 lysogenization of strain KS 463  site specific integration of  DE3 prophage into an E. coli chromosome-  lysogenization with a three-way infection with  DE3, the Helper phage (B10) and a selection phage (B482).  most of the surviving cells are usually  DE3 lysogens  DE3 was made by inserting the T7 RNA polymerase gene behind the lacUV5 promoter into the BamH I cloning site of  D69 (Studier and Moffatt(1986) J. Mol. Biol. 189, ).

3 M. HAERTLEIN, INSTITUT MAX VON LAUE - PAUL LANGEVIN 3 rd D-Lab Meeting, 28 th April 2003 EMBL Heidelberg Plasmid for the rat  C -crystallin (Prof. N. Lubsen,Nijmegen, Netherlands) with a His tag E.coli strain KS 463 (Genetic Stock Center, Yale University) auxotrophic for Trp was lysogenized with  DE3 Fed batch culture in fermentor H Trp used until density OD 600 = 6 Rifampicin added to improve solubility Expression

4 M. HAERTLEIN, INSTITUT MAX VON LAUE - PAUL LANGEVIN 3 rd D-Lab Meeting, 28 th April 2003 EMBL Heidelberg Purification Purified on an Immobilized Metal Affinity Column (Cobalt) and eluted in the presence of imidazol. His tag removed with thrombin Concentrated on Millipore concentrating device Gel filtration Yield 60 mg from 20g of cells Stored at -20C

5 M. HAERTLEIN, INSTITUT MAX VON LAUE - PAUL LANGEVIN 3 rd D-Lab Meeting, 28 th April 2003 EMBL Heidelberg H-  C crystallin  Mass = D-  C crystallin  Mass = Mass spec results


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