Protein Expression Facility (PEF)

The Protein Expression Facility (PEF) aims to provide a high quality recombinant expression service, principally to academic research groups based at the University of Birmingham.

The general principles of the PEF are:

  • to provide a service as close to cost price as possible, but consistent with sustainability of the facility
  • to cater for production of a wide variety of proteins
  • to enable a wide range of downstream uses
  • to be responsive to customer needs where possible

A final aim is that the PEF acts as a focal point for advice about all aspects of recombinant protein production and downstream applications.

Planning expression constructs
The PEF offers free advice regarding design of recombinant expression constructs, and will work with you in order to design optimal constructs for whatever expression system or downstream application you are interested in. If you are interested in discussing a new project, please email the PEF at: pef@contacts.bham.ac.uk

Molecular Cloning Services...

The PEF offers molecular cloning services including construct design, DNA primer design and synthesis, and molecular cloning of constructs in the expression vector of choice. Also for certain complex cloning requirements, the PEF can advise on use of commercial cloning services. So if you want to generate recombinant expression constructs but don't have good molecular cloning skills within your group, or just want to kick-start a new project area, then the PEF will help you get your project started.

Protein Expression Services and Systems...

The PEF offers protein expression services focused on three recombinant expression systems, which collectively will enable production of a broad range of target proteins suitable for a wide variety of downstream applications. These are E.coli, mammalian cell expression, and insect cell expression. In each case, the PEF offers small scale test expression, expression optimisation, and large scale expression.

E.coli: A widely used expression system, E.coli production is quick, highly manipulable, low-cost in terms of reagents, and capable of generating high expression of the protein of interest. In addition, a diverse range of expression vectors and affinity tags/fusion partners are available, and efficient 2-H, 13-C, 15-N and Selenomethionine labeling approaches are established for downstream NMR and X-ray crystallographic applications. Limitations of E.coli expression systems include lack of post-translational modifications, and absence of suitable chaperones necessary for folding of some eukaryotic proteins.

Mammalian 293T: Recombinant expression in the Human Embryonic Kidney (HEK) 293T variant cell line is a widely used, quick and relatively cheap approach that is capable of generating high levels of a wide range of eukaryotic target proteins. Advantages include incorporation of human post-translational modifications, availability of glycosylation mutant strains, and the fact Selenomethionine labeling strategies for X-ray crystallographic approaches are established in this system. In addition, 293T expression is widely used for generation of immunogens for antibody production.

Insect: Stable transfection of Drosophila S2 cells is a well-established system for recombinant production of a wide range of proteins, including oligomeric complexes and cell surface proteins, and allows good expression levels and relatively homogenous glycosylation. One limitation is the length of time (~4 weeks) for selection of stable expression clones.

Protein Purification...

The PEF will offer advice concerning protein purification steps, and where possible, will offer protein purification services. Some purification services, such as GST or His-tag purifications, will be offered as a standard service, whereas less standard procedures will require discussion with PEF personnel and will be arranged on a case-by-case basis.

The protein expression systems used by the PEF will enable a wide range of downstream applications for recombinant proteins generated. Some of these are listed below, with relevant UofB research facility details provided.

1. Use as immunogens CMDS Antibody Production Facility Contact: Dr Margaret Goodall

2. Cell staining of functional reagents CMDS Flow cytometry facility Contact: Dr Matt MacKenzie

3. Biophysical Studies (eg Circular Dichroism, Analytical Ultracentrifugation, Isothermal Titration Calorimetry) Birmingham Biophysical Characterization Facility (College of LES) Contact: Professor Timothy Dafforn, Professor Ben Willcox

4. Nuclear Magnetic Resonance Henry Wellcome Building for Biomolecular NMR Contact: Professor Ulrich Gunther

5. X-ray crystallography Birmingham X-ray Diffraction Facility (College of LES) Contact: Dr Fiyaz Mohammed, Dr Klaus Futterer

BMG PHERAstar FS with micro injectors...

The PHERAstar FS microtiter plate reader is located in room 408 of the Cancer Sciences Laboratory. It was bought in 2013 through a joint collaboration from colleagues in CMDS and then made available to all CMDS Researchers in late 2013.

A small service charge per group is administered by the HUB to cover the cost of the annual service. Access to the PHERAstar FS is administered by Jamie Webster and booking to use this equipment is via Stratocore.

Further help is provided by BMG Labtech and they service the machine annually. If you need further details please contact Jamie Webster J.R.M.Webster@bham.ac.uk

The PHERAstar® FS is the next generation microplate reader for High-Throughput screening, specifically conceived by BMG LABTECH for highest sensitivity and speed. Its unique features make it superior to any other microplate reader currently on the market. German engineering with the latest technology makes the PHERAstar FS the Gold Standard for HTS. It performs all the leading non-isotopic detection technologies such as:

  • Ultra-fast UV/Vis Absorbance Spectra
  • Fluorescence intensity (including FRET)
  • Fluorescence polarization/Anisotropy
  • Time-Resolved Fluorescence, including TR-FRET
  • High-end AlphaScreen®/AlphaLISA®
  • Luminescence (flash and glow), including BRET

Sequential Dual Excitation, Simultaneous Dual Emission and ratio metric calculations are just some of the key features of the PHERAstar FS for multi-chromatic applications such as FRET, TR-FRET, BRET and FP.

Application specific Optic Modules can be activated by a simple mouse click, and all the necessary components are selected automatically. Assay flexibility is enhanced by top / bottom reading, onboard reagent injectors, precise temperature control and multi-mode shaking capabilities.

The PHERAstar FS provides uncompromised sensitivity, speed and dynamic range in plate formats up to 3456.

Figure 1 any plate format up to 3456

Figure 1 any plate format up to 3456

Tandem Technology

The PHERAstar FS incorporates BMG LABTECH’s unique Tandem Technology, which is based on two technological concepts: an ultra-fast UV/Vis spectrometer for absorbance and high-performance optical filters for all other detection modes.

Innovative Optical Design

The outstanding sensitivity of the PHERAstar FS is based on a new, innovative lens-based optical design which is composed of three independent light sources, Simultaneous Dual Emission detection, and software controlled top / bottom reading. Depending on the application, users can choose one of the following light sources:

  • High energy xenon flash lamp
  • Nitrogen laser for TRF / TR-FRET
  • Solid-state laser for AlphaScreen® /AlphaLISA®

Two matched pairs of photomultiplier tubes (PMTs) are used in the PHERAstar FS. The first pair of PMTs is for simultaneous luminescence and fluorescence detection, and the second pair of PMTs detects time-resolved fluorescence based assays. Distinct advantages of the nitrogen laser include increased assay window and the “flying mode” measurement for TR-FRET based assays. A single laser flash excites the donor molecules sufficiently so that measurement in a well takes place without stopping plate movement. Thus reading time for an entire plate is significantly decreased.

Simultaneous Dual Emission

BMG LABTECH pioneered the technique of Simultaneous Dual Emission detection for microplate readers. Because numerous assays require detection of two wavelengths, Simultaneous Dual Emission offers a significant advantage by cutting read times in half. It corrects flash-to-flash variations, assay effects such as photo bleaching, decaying kinetic signals, or fluctuating conditions like temperature, following Optic modules which enable accurate measurement of: pH, and evaporation. Simultaneous Dual Emission detection can be used in any assay that measures two wavelengths or polarization vectors, including FP, FRET, and HTRF.

schematic

Figure 2 Schematic layout of the Simultaneous Dual Emission (SDE) optical

Optic Modules

The assay specific Optic Modules are the core elements of the PHERAstar FS. They contain all the necessary optical components including excitation and emission filters, dichroic mirrors, beam splitters and polarization filters. The PHERAstar FS can accommodate up to six Optic Modules and the user can easily add or replace them within seconds. All Optic Modules are barcoded, and the PHERAstar FS automatically selects the proper module for your assay.

The CMDS PHERAstar FS has the following capabilities:

 

Filter

Code

excitation wavelengths

maximum variation

emission wavelength

maximum variation

1

HTRF

1209D1

337

plus/minus 10

665 620

plus/minus 10

2

FI

1209B1

485

plus/minus 10

520

plus/minus 10

3

FI

1311A1

540

plus/minus 10

590

plus/minus 10

4

ALPHA SCREEN

1211B1

680

plus/minus 10

570

plus/minus 10

5

FI

1205A1

280

plus/minus 10

340

plus/minus 10

6

TRF

1201B1

337

plus/minus 10

615

plus/minus 10

7

BRET 2 PLUS

1302A1

515-30

plus/minus 10

410-80

plus/minus 10

8

FP

1107B1

485

plus/minus 10

520 520

plus/minus 10

The machine has built in UV/Vis absorbance plus chemiluminescence and also has the same capability of the Nano drop when using the L VIS plate S/N 680-0287 with the option to screen 14 samples at one go.

Smart Reagent Injection

Many popular assays require the ability to monitor a signal before, during and after the addition of a reagent to a well. The two onboard Reagent Injectors are ideally positioned so reagents can be added to the well currently being read. Thus, concurrent reading and injection ensures that even the initial part of fast kinetic reactions can be monitored. The injectors are readily accessible and are housed within the instrument to safeguard any light sensitive reagents.

An exceptionally small dead volume and back flushing ensure that precious reagents are conserved. Users can tune all parameters, such as plate shaking, injection speed, timing, and the number of injections per well. Delivery volumes are adjustable for each well, allowing users to automatically produce dilution schemes and concentration gradients across the microplate.

onboard-reagent-injectors

Figure 3 Onboard Reagent Injectors for simultaneous injection and reading.

Injectors were factory installed with this machine and are checked yearly for accuracy in delivery.

Facility Equipment (located in Robert Aitken Building)...

The following equipment is available to use in the facility please arrange booking access to facility and training with Jamie Webster prior to use.

G13

3x New Brunswick INNOVA 44R shaking incubator

1

  • Large easy to read displays shows set point and actual values
  • Integral timer, 0,1 to 99,9 h, shows elapsed time or counts down, automatic switch-off and audible alarm when program is complete. Induction experiments can be controlled via the programing so that you can induce during the day restrict induction time between 1-16 hours overnight and then reduce  temperature to 4oC with minimal shaking (25rpm) and harvest the next morning.
  • Microprocessor controller maintains precise temperature, stores programs for repeat use and recalls last set points, even when unit has been shut off. Will also restart if power is interrupted
  • Triple eccentric counterbalanced drive with brushless motor ensures uniform motion regardless of where the sample is on the platform even when fully loaded and operating at top speed
  • Air circulation system ensures rapid temperature equilibration and uniformity, multi-function reservoir humidifies the chamber to minimise sample evaporation

Incubator 1
is configured for overnights small cultures on sticky mats and microplate work using a universal platform

Incubator 2
is configured for 5 litre flasks and small flasks using a universal platform

Incubator 3
is configured for 12x 2 litre flasks 

2x Beckman Avanti J-26 XP High performance Centrifuge

2

  • Fully compatible with the J-Lite rotors series
  • Fully adaptable with more than 32 rotor combinations to meet a wide range of separation needs (fixed angle and swinging bucket options ranging frin 43.2 mL to 6 L capacity.
  • Exclusive SR Drive Technology with definable accel and decel rates improve efficiency, yield, and confidence

Rotors available (users are required to provide their own bottles and cap assemblies)

3J-LITE® JLA-8.1000 Rotor, Fixed Angle, Aluminium, 6 x 1,000 mL, 8,000 rpm, 15,970 x g

 

 

 

4JA-25.50 Rotor, Fixed Angle, Aluminium, Biosafety Lid, 8 x 50 mL, 25,000 rpm, 75,600 x g

 

 

 

G14

2x New Brunswick Galaxy S Series 170 S CO2 Incubator

  • Gentle, fanless convection circulation allows full use of the incubator interior
  • IR CO₂ sensor with automatic auto-zero to ensure accurate calibrated measurements
  • Galaxy 170 S is available with optional high temperature disinfection (at 120 °C)
  • Stackable - two units high with optional stacking kit.
  • Connected to C02
  • Set at 37oC with 5% C02

2x New Brunswick INNOVA 44R shaking incubator for S2 / S9 work

  • Same as the incubators in G13 however the setup is for 27oC with 17 rpm shaking on a Universal platform without clips.
  • Non-CO2

1x Wheaton roll-in incubator for cell production

  • This can take up to 55 2 litre roller bottles. Set at 37o
  • Non-CO2

5

G22 RAICR

ÄKTA pure 25 L

6

Specifications:

System pumps Two bio-inert high performance dual piston pumps for flow rates up to 25 ml/min and pressure up to 20 MPa Pressure sensors One pressure sensor placed after system pump Mixer Gradient mixer with 1.4 ml chamber included. Injection valve Connections for sample application from both loop and sample pump in one valve UV monitor (U9-L, Fixed Wavelength) Detect proteins at 280 nm using long-life LED technology. Can also be used as a second UV monitor. Conductivity monitor Wide-range (0.01 to 999.99 mS/cm), for gradient verification in all common chromatography techniques.

Outlet Valve Kit (V9-Os, 1-outlet) Switch between fraction collector, one outlet position and waste.

Inlet Valve Kit (V9-IAB) Switch between multiple buffers and cleaning solutions. Selection between 2 A and 2 B inlets in a single valve.

Fraction collector F9-C Covered cassette format x-y fraction collector for safe collection of up to 576 fractions in deep-well plates, tubes, and bottles.

Air sensor L9-1.5mm Automatically detect air in inlet tubing, e.g. in order to pause system if running out of buffer or for complete loading of sample. Can be attached to system using adapter for air sensor and bottle holder. Uses 1/8 inch connectors. For use with ÄKTA avant or ÄKTA pure.

Loop Valve Kit (V9-L) Automate sample application from up to five sample loops or collect intermediate fractions in automated two-step purification.

Column Valve Kit (V9-C, 5-columns) Switch automatically between 5 columns. Automate cleaning and increase eluted concentration using column bypass and reverse flow. Safeguard columns through pre-and post-column pressure measurements.

Column holder for columns with outer diameter between 10 and 50 mm. Attached to ÄKTA Avant or ÄKTA pure using the rail system.

ÄKTA purifier UPC10

7

ÄKTA purifier core systems are versatile, modular liquid chromatography systems for fast and reliable separations of proteins, peptides, and nucleic acids by FPLC. The four available core systems can be combined with ÄKTA design automation kits and hardware to achieve tailor-made solutions for your protein purification needs.

Using ÄKTA design automation kits to expand ÄKTA purifier core systems also eliminates a number of common problems in FPLC purification, such as long sample loading time, frequent column cleaning, and air corrupting thesystem.

• Four core system configurations for greater versatility

• High-performance purification by FPLC

• System configurations designed to meet individual

research needs

• ÄKTAdesign automation kits can be added to provide greater flexibility

  • Wide range of purification scales

ÄKTA purifier 10 and UPC 10 configurations are designed for purification of microgram to milligram quantities of protein, operating at flow rates of 0.001 to 10 ml/min at pressures of 0 to 25 MPa.

Columns available for use on both ÄKTA’s:

Mono Q 4.6/100 PE

Mono Q is a strong anion exchanger prepacked with MonoBeads in a Tricorn column. The column is an excellent choice for small-scale polishing in purification of proteins, peptides, and other biomolecules when high purity is required.

Mono S 4.6/100 PE

  • Mono S is a strong cation exchanger prepacked with MonoBeads in a Tricorn column. The column is an excellent choice for small-scale polishing in purification of proteins, peptides, and other biomolecules when high purity is required. The column has 1.7 ml bed volume for small-scale purifications.
  • Both share the following specifications:
  • Monodisperse 10 µm porous beads yield high resolution, dynamic capacity, reproducibility, and durability.
  • Maximum resolution is achieved from the efficiency of small, perfectly spherical monodispersed particles optimally packed in columns coupled with the excellent selectivity’s of the Q ion exchangers.
  • Compatible with ÄKTA design, FPLC System, and other high-performance LC systems.
  • MonoBeads are also available as Mono P for chromatofocusing
  • The Tricorn columns offer high-resolution separations with greater loading capacity but slightly lower resolution than MiniBeads columns.

RESOURCE Q

  • Resource Q columns are prepacked with Source 15Q, a strong anion exchanger for intermediate lab-scale purification and large-scale polishing of biomolecules.

RESOURCE S

  • Resource S columns are prepacked with Source 15S, a strong cation exchanger for intermediate lab-scale purification and large-scale polishing of biomolecules with ion exchange chromatography.
  • Both share the following specifications:
  • SOURCE 15Q and 15S media are highly suitable for fast, high-resolution purifications and easy scale-up.
  • Rigid, monodispersed, spherical particles with controlled pore-size distribution offer excellent flow characteristics, sample loading of up to 25 mg of protein/ml, and improved stability in organic solvents and pH extremes
  • RESOURCE prepacked columns for fast separation. Separation times using 1 ml RESOURCE columns are less than 3 min at 9.6 ml/min and about 20 min using a peristaltic pump at 1 ml/min.
  • Improved capacity compared to MonoBeads but with a slightly lower resolution.

HiLoad 26/600 Superdex 200 pg

HiLoad 26/600 Superdex 200 prep grade are prepacked XK columns designed for high-resolution preparative gel filtration chromatography. The column is an excellent choice highly suitable for separating MAbs from dimers and contaminants.

HiLoad 26/600 Superdex 75 pg

HiLoad Superdex 75 prep grade are prepacked XK columns designed for high-resolution preparative gel filtration chromatography separating proteins and polynucleotides.

Both share the following specifications:

  • High resolution with short run times and good recovery.
  • High capacity at high flow rates.
  • Separations can be scaled up to production levels.
  • Prepacked for convenience and reproducibility.
  • 1/16” fittings for convenient connections to ÄKTA design systems.

Superdex prep grade (pg) is produced by covalent bonding of dextran to highly cross-linked agarose. The separation properties of these media are determined by the dextran component. Steep selectivity curves give excellent resolving power for peptides and proteins in the molecular weight ranges, Mr < 10 000 (Superdex 30 pg), Mr 3 000 to 70 000 (Superdex 75 pg), and Mr 10 000 600 000 (Superdex 200 pg). The medium combines high mechanical strength with high hydrophilicity, allowing high flow rates, and minimal nonspecific interaction.

Superdex 200 10/300 GL

  • High-resolution separations of proteins, peptides, and other biomolecules according to size.
  • Highly suitable for the polishing step in a purification procedure.
  • Easy and predictable scale-up.
  • Excellent reproducibility and durability.
  • For rapid protein homogeneity analysis or purity check use the 5/150 GL short columns.

Training...

The PEF can offer training to internal UoB staff and UoB Postgraduate/Postdoctoral colleagues.

We can provide training in various aspects of recombinant protein production as a charged service.

Areas covered include molecular cloning techniques, plasmid design, protein expression (in E.coli, 293T and Drosophila expression systems), refolding procedures, use of the AKTA purifier or pure and protein purification steps.

If you are interested in exploring training options, please contact Jamie Webster