Transcript Points for Discussion

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
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
PSI Program
is Making an
Impact
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
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, Nustag, S-tag, your favorite tag etc
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
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
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Protein evolution - split GFP assay
Limited proteolysis/MS
Use of orthologues
Expression of protein pairs
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
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
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
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
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?
Points for Discussion (cont.)
• Comments, feedback, suggestions
• Future meetings