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

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 Development of high throughput methods for membrane associated proteins expands the Target groups Informatics and expression tools HT methods/screens NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

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 Development of high throughput methods for membrane associated proteins expands the NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

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

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

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

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

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

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

Soluble domains – multiple TMH 6+-MSH   APC1910 19 11 4 6 12 11 8 19 4 4 139 4 6 175 APC1932 113 4 6 81 4 19 30 14 19 APC1917 134 9 14 132 19 19 1 19 27 6 20 35 APC1979 131 20 12 37 31 19 14 25 19 APC1918 135 9 14 131 19 19 1 19 27 6 20 35 APC1988 39 4 3 39 3 4 155 20 4 6 12 11 9 APC1926 140 24 3 24 28 12 6 APC2003 130 19 34 29 12 8 APC1930 144 11 6 341 6 6 3 23 25 4 24 16 APC2011 14 9 47 44 230 12 6 6 18 3-MSH   4 and 5-MSH APC1902 20 160 3 APC2012 138 20 4 3 APC1938 129 19 20 15 APC2018 4 14 4 210 APC2064 124 14 6 167 APC1901 146 27 147 7 APC2068 12 162 5 APC1946 157 14 30 17 6 4 APC2079 126 20 31 APC1968 8 6 161 3 14 NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

Eukaryotic proteins - Domain scanning approach Informatic analysis 1500 1600 1800 1700 100 Automated design component Outline of Domain screening process with preliminary data from BRCA1 screen HT screen NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

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

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

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

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

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

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