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Expression of secretory/periplasmic proteins and “soluble” domains of membrane proteins

Expression of secretory/periplasmic proteins and “soluble” domains of membrane proteins. Frank R. Collart Midwest Center for Structural Genomics Biosciences Division Argonne National Laboratory. Goal.

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Expression of secretory/periplasmic proteins and “soluble” domains of membrane proteins

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  1. Expression of secretory/periplasmic proteins and “soluble” domains of membrane proteins Frank R. Collart Midwest Center for Structural Genomics Biosciences Division Argonne National Laboratory NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  2. Goal • Goal:Evaluate high throughput methods for the cloning, expression and solubility analysis of secretory/periplasmic proteins and soluble domains of membrane proteins. • Evaluate success rate from expression to crystal structure • Identify obstacles or components that require improvement • Determine suitability for incorporation into the MCSG pipeline Helical membrane proteins Secretory and periplasmic proteins Oligo program Target groups Informatics and expression tools HT methods/screens NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  3. Experimental Approach Bacillus subtilis – domain set • SignalP used to identify signal sequences • First 70 amino acids • HMM and NN options • TMHMM for membranes spanning segments • Filtered for size and methionine content • 205 targets. • First pass (pMCSG7 vector) • Standard HT methods used for cytoplasmic targets NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  4. Soluble clone descriptions NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  5. Summary of expression experiments Total 5+ MSH 4 MSH 3 MSH 2 MSH 1 MSH N-term S/A NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  6. Solubility distribution based on target class and topology Classification NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  7. Results Summary NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  8. APC1972 model NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  9. Soluble domains Topology cartoons for targets with 2-MSH NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  10. Soluble domains – multiple TMH NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  11. 100 1500 1600 1800 1700 Eukaryotic proteins - Domain scanning approach Informatic analysis Automated design component HT screen NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  12. Domain primer tool NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  13. Domain permutations expressed in E. coli Tag detection-Expression Target plate map Tag detection-Solubility NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  14. Human endothelial cell protein MRSL66 660 amino acids Membrane anchor Domain 1 Domain 2- putative Glycosyltransferase IKLILDTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGYGSVCS IKLILDTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGY IKLILDTRRAISEANEDPEP VEAECHWADTELNRRRRRFCS IKLILDTRRAISEANEDPEP VEAECHWADTELNRRR IKLILDTRRAISEANEDPEP VEAECHWADTE ---------DTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGYGSVCS ---------DTRRAISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGY ---------DTRRAISEANEDPEP VEAECHWADTELNRRRRRFCS ---------DTRRAISEANEDPEP VEAECHWADTELNRRR ---------DTRRAISEANEDPEP VEAECHWADTE ------------------- ISEANEDPEP VEAECHWADTELNRRRRRFCSKVEGYGSVCS NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  15. Solubility analysis of MRSL66 domain permutations Domain permutations Set 3 Set 2 Set 1 -IKLILDTRRAISEANEDPEP Set 1 - DTRRAISEANEDPEP Set 2 -ISEANEDPEP Set 3 NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  16. Summary • Secretory/periplasmic proteins and low complexity helical membrane protein are targets for SG pipelines. • Optimization • Improved methods for prediction of signal sequences and the boundaries of the soluble domains of helical membrane proteins (i.e. specifically determine the N- and C-terminus of the domains) • Tool development • Need to evaluate alternative strategies such as periplasmic expression systems or the incorporation of fusion tags to enhance. NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

  17. Acknowledgements Andrzej Joachimiak PI, MCSG Shiu Moy Cloning/expression Denise Holzle Cloning/expression Natalie Maltsev CMT/ANL Gong Xi Yu CMT/ANL Lynda Dieckman BIO/ANL Diane Rodi BIO/ANL The MCSG team NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

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