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
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

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
Development of high throughput methods for membrane associated proteins expands the
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. Topology cartoons for targets with 2-MSH
Soluble domains
NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

10. Soluble domains – multiple TMH
6+-MSH
APC1910
APC1932
APC1917
APC1979
APC1918
APC1988
APC1926
APC2003
APC1930
APC2011
3-MSH
4 and 5-MSH
APC1902
20 160 3
APC2012
138 20
APC1938
20 15
APC2018
4 14
4 210
APC2064
124 14
6 167
APC1901
146 27
147 7
APC2068
12 162 5
APC1946
APC2079
126 20 31
APC1968
3 14
NIGMS PSI Protein Production and Crystallization Workshop, March 28, 2004

11. 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

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
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

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