1. 2004 PP&CW Optimization of protein expression and solubility Alternative and novel prokaryotic expression systems Eukaryotic expression systems Methods to minimize sample heterogeneity and improve crystal diffraction Membrane associated proteins

2. 2004 PP&CW Major progress in technology development in protein expression, purification and crystallization Protein structure determination pipelines in place that are capable of producing significant number of novel protein structures – supported by automation Robotic platforms for cloning, purification and crystallization established “Low vs high hanging” fruit issues

3. PSI Program is Making an Impact

4. Points for Discussion (cont.) At present majority of protein production is done in E. coli Bottlenecks to efficient protein expression in E. coli –Inefficient transcription – optimizing mRNA (synthetic genes) –Inefficient translation – optimizing codon usage, alternative expression of rare tRNAs (pRARE, Magic etc) –Inefficient folding - approaches to refolding proteins Use of chaperones, cofactors in vivo and in vitro Designing a chaperonin/osmolyte folding array systems Role of osmolates on protein stability and crystallization –Protein solubility issues – new approaches –Screening for “good” protein expression systems and scale up problems

5. Points for Discussion (cont.) We need to develop a flexible cloning strategy – E. coli, yeast (Pichia pastoris), insect cells/baculovirus, eukaryotic cells, cell free expression Different vector/host combinations Protein co-expression - improves expression and solubility Fusion protein expression- issues of metabolic cost Affinity tags – there is no magic tag –N- vs C-ternimal tag, His-tag, Trx-tag, GST-tag, Nus- tag, S-tag, your favorite tag etc

6. Points for Discussion (cont.) A significant fraction of targeted proteins are “left behind” This workshop showed that there is still lots of room for improvement We need a strategy for higher output Many questions remain unanswered –How much expression is effected by the origin of the protein? –How much host can be tune-up to improve expression? –Are there specific cofactors/helpers needed? Examples of advanced metabolic engineering showed that we can make protein expression better

7. Protein solubility Very difficult to predict solubility of proteins in E. coli –On-column chemical refolding of proteins from inclusion bodies Protein super-chunking and domain identification –Computational predictions not very reliable Domain functional inference Sequence analysis - domain parsing –Experimental approaches Protein evolution - split GFP assay Limited proteolysis/MS Use of orthologues Expression of protein pairs

8. Protein purification Protein production and purification, parallel approaches to purification One-step purification and processing of fusion proteins –Application of engineered subtilisin –TEV protease on column cleavage New HTP robotic platforms for protein purification

9. High value targets – what to do? Lowering protein surface entropy to enhance crystallization - rational surface mutagenesis High affinity single-chain antibodies for structural genomics Eukaryotic expression technologies –Transient transfection into eukaryotic cells: an alternative to bacterial and insect cell system Cell free expression system in wheat germ system for NMR and X-ray crystallography as an alternative – new robotic system Issues post-translational modification –Producing recombinant glycoproteins in the baculovirus-insect cell system How make these approaches HTP

10. Protein Crystallization Issues Protein sample homogeneity and quality control High throughput screening to determine lead crystallization conditions A microfluidic system for protein crystallization using nanoliter volumes Crystal salvaging efforts –New crystallization solubility screens Database mining - correlation of protein properties and crystallization conditions with crystallization success High-throughput capillary-based crystallography Growing larger protein crystals

11. Membrane Proteins (IMP) Successful expression of functional membrane proteins in E. coli and other systems (rather than expression of frustration of the investigator) Automation of large-scale purification of membrane proteins Obtaining diffraction quality crystals of IMPs –Use of thermophilic sources –Ligands for soluble domains (antibody vs natural ligands) Genomics approach to expression of membrane proteins (bacterial and eukaryotic) Expression of periplasmic domains and a soluble domains of membrane proteins in E. coli is possible using HTP Use of NMR for small proteins and solid state NMR

12. Databases Integration of pipelines with LIMs and databases How to capture all relevant data Database mining to improve the process –Access to databases Sharing data issues How the structural data can be used for modeling, improve quality of models?

13. Points for Discussion (cont.) Comments, feedback, suggestions Future meetings