Shared Facilities: Protein Expression Facility
John O. Trent, co-Director
Andrew N. Lane, Director
Scott Adcock, Technician
Room 4A JG Brown Cancer Center
429 S. Jackson St.
Contacts for scheduling, advice and information:
General laboratory information, time schedules, fee structures.
Scott Adcock - rsadco03@louiville.edu or 502-852-4437
Advice on procedures and approaches; stable isotope labeling
Andrew Lane -
anlane01@louisville.edu or 502-852-3067
Chemically oriented questions.
John Trent- jotren01@louisville.edu
Overview
Biophysical techniques including crystallography, NMR, calorimetry need substantial
quantities of pure protein (typically ≈ 10 mg or more). This is
much larger than usually generated in biological laboratories, and requires
more than simple scale up of facilities.
- Many of these methods also require incorporation of stable isotopically
labeled residues or atoms. The relative expense places efficiency
at a premium. One way of allaying costs is one of scale; bulk buying
reduces per unit costs.
- Infrequent producers of a protein for structural and biophysical analysis
may not wish to convert their laboratory to a production factory, and then
have specialized equipment sitting idle for long periods.
- Efficient large scale production of pure proteins often needs different
approaches and therefore expertise
- Solutions: centralized facility that maintains appropriate equipment for
cell growth, protein expression and purification. Such a facility can
also offer expertise and assistance, all at cost.
Facilities
The Laboratory was established in Spring, 2004, and currently has the following facilities:
Microbiology/fermentation:
- Class II Microbiological hood
- Autoclave (accepts up to 6L flasks)
- NewBrunswick Orbital shaker (up to 6L flasks)
- NewBrunswick Bioflow3000 with 3L jar
- Sorvall Evolution preparative centrifuge with Fiberlite 3L and 8x50 ml rotors
Purification:
- Cold cabinet (4°C) housing an Aktapurifier FPLC system: numerous columns
(affinity, ion exchange, hydrophobic, sizing, desalting, chromatofocusing)
Varian Prostar ternary gradient HPLC system
General:
- Barnstead water purifier (18 Mohm, uv lamp and 0.2 mm filtered-RNase free water)
- MJ Research PCR machine
- Eppendorf microfuge
- Accumet pH/conductivity meter
- Balances etc.
- Electrophoresis
- Freezer (-20°C)
- Chemical hood w/ solvent and acids cabinets
- Basic reagents, Kits etc.
Capabilities
- Transformations of E.coli or P. pastoris
- Small and large-scale growths in rich (LB, TB etc) media
- Growth in defined media (e.g. for isotope labeling)
- Cell harvesting
- Cell lysis (chemical or mechanical)
- FPLC/HPLC purification by affinity chromatography, ion exchange and gel permeation. Reverse phase by HPLC.
- Purity assessment by SDS-PAGE.
- Determination of the absorption coefficient at 280 nm.
- Advice
Services
Facility costs: equipment maintenance, technician, general supplies, bulk
orders (savings passed on to the user).
Recharge to recover some of the fixed costs and running costs of projects.
Rates: depend on level of service
Full service: From plasmid to protein (no guarantees)
Partial services: e.g. producing an inoculum and cell paste
from 1-5 L culture, protein characterization.
Do it yourself (subject to training)
Isotope labeling:
The costs of the stable isotopes need to be covered. Current rates are
(e.g.) $30-35 / g 15N ammonium sulfate (1.5 g/L), $130 /g [U-13C6] glucose
(2-4 g/L), $350/kg D2O. These are bulk purchase rates, and they vary as a complex
function of time.
Fees
Rates: Full service based on no. L culture grown and time taken for purification.
Cost for:
Media: including IPTG etc.
Transformation: competent cells, plasmid isolation
Protein purification:
1. technician assisted- 10-20 g cell paste level. Lysis +first column, $150,
subsequent columns @ $100
2. No assistance, using core materials and facilities: $50 set up plus $5/h
Quality control:
SDS PAGE (Atto mini gel): making gel, preparing sample, running with standard,
staining and documentation:
UV-vis spectrum and absorption coefficient-sample prep and analysis $40
Example protein preparation: E.coli cell transformation, unlabeled media 4L
scale, growth and induction, harvesting, lysis, 1 column preparation and QC
ca. $600
Exact costs to be established
Planned expansion of capabilities
In vitro translation:
- E. coli lysate with dialysis system (T7 polymerase co-translation)
- Can do mg level
- Develop wheat germ lysate for eukaryotic protein expression
- Other lysate systems
Suspension mammalian cell culture:
- e.g. production of membrane proteins (e.g. B cells)
- controlled growth conditions for metabolomics
- e.g. hematopoietic cells
- adherent cells on beads grown in suspension
Intein technology:
- domain splicing/segmental labeling
Ongoing Projects
vMIP-II: expression and purification of 13C-labeled protein from P. pastoris
for NMR relaxation analysis (A.N. Lane)
Human SDF-1a expression and purification from E.coli for biophysical
and cell biology analyses. This protein is available to researchers at
cost. (A.N. Lane, M.Z. Ratajczak)
Nucleolin domains expression and purification of unlabeled and 13C/15N labeled
protein in E.coli for NMR analysis and biophysical characterization (J.O. Trent)
Thrombin expression and purification in E. coli for NMR (M. Maurer)
MBP1/enolase expression and purification for biophysical characterization
(D. M. Miller)