The Protein Production and Analysis core facility offers protein expression services based on three recombinant platforms consisting of mammalian, microbial and insect based systems. In each case a small scale test expression, expression optimization, and large scale expression package can be provided. The primary expression systems used are E.Coli, suspension based CHO or HEK293, and Sf9 insect cells.
With the ability to utilize three platforms, the Core can produce a broad range of proteins for a variety of downstream applications with operations going from research lab based protein yields to scaled up protein production yields using pilot scale bioreactor/fermenter systems.
With the implementation of advanced biochemical engineering techniques protein yields can be significantly increased.
Our analytical services are focused on biophysical characterization of structural and functional properties of proteins using several methods, including the latest Surface Plasmon Resonance (SPR) techniques. We can perform quality control of protein samples and measure protein-protein interactions.
The protein production and analysis core provides a range of services to all SBP researchers and to external users such as custom plasmid preps and protein expression optimization in the desired expression system.
The facility has the ability to produce secreted and intracellular proteins including baculovirus stock production and amplification of master stocks.
If a service that is required is not listed please contact us as we have the resources to take on custom projects.
The mammalian system can produce biological active forms of protein with proper folding and post translational modifications including glycosylation.
Two good platforms to use for mammalian glycoprotein production are are CHO and HEK293 cells. CHO cells are known to be robust in culture and are able to produce a variety of recombinant glycoproteins at high levels on a large scale. This platform has even gained acceptance as the gold standard in biopharmaceutical production.
Our expertise can get your glycoprotein of interest produced in the minimal amount of time at milligram or gram* quantities in conjunction with protein expression optimisation projects if need be.
*May require stable cell line generation for increased amount of glycoprotein expression.
The use of E.coli as a production platform has the advantages of being quick, low-cost, in comparison to other systems, and capable of generating high expression levels of the protein of interest. Due to its popular use, a wide range of expressiom vectors and affinity tags are also available for this platform.
This expression system lacks post translational modifcations and the necessary chaperones required for some eukaryotic proteins. In many cases the expressed protein is insoluble and accumulates in inclusion bodies. This can most often occur under conditions of high level protein expression.
For this system we offer a variety of scales from milliters of culture to a maximum of 50L per run. The choice of scale will depend on many factors such as the customers needs, expression trials, amount of protein desired, and protein stability, to name a few.
We also offer the baculovirus system as a rapid way to produce glycosylated protein. The main system we use for this is the Bac-to-Bac® Baculovirus Expression System which is faster and easier because the technology relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells, which often results in low titers.
Baculoviruses are rod-shaped dsDNA viruses mainly found in insects. The most common baculovirus used for expression studies is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which relies on the lepidopteran species Spodoptera frugiperda (Sf cells) and Trichoplusia ni (High Five™ cells) as host insects. AcMNPV particles surround themselves with a protective matrix consisting of the protein polyhedrin. Polyhedrin is expressed at extremely high levels (up to 50% of all cellular protein) at the end of the baculovirus life cycle due to an extremely strong promoter pPolh. The baculovirus expression system makes use of the fact that in cell culture a polyhedrin coat is not essential for virus propagation and thus heterologous proteins can be expressed under the control of the pPolh promoter. Furthernmore, inefficient secretion from insect cells may be circumvented by the addition of insect secretion signals (eg. honeybee melittin sequence).
Depending on the protein of interest this can be a good candidate, depending on expression, since potential N-linked glycosylation sites are often either fully glycosylated or not glycosylated at all, as opposed to expression of various glycoforms that may occur in mammalian cells.
Typical turnaround time:
- Cloning: 2-3 weeks
- Expression evaluation: 5 to 6 weeks
- Production and purification: 1 to 4 weeks
We can provide custom vector design as part of the cloning service which includes vectors for particular expression platform services we carry. The cloning process can sometimes be difficult and time-consuming, especially if you need to create custom vectors to express proteins using multiple systems. Customers can provide cDNA of their expression target or we can order and verfiy a sequence of interest. In a typical project the gene of interest can be amplified from a customer-supplies vector by PCR and clones into a specific required expression vector. Afterwards, the expression construct will be sequenced for quality control.
A typical cloning project can take 2-3 weeks.
Purification & Analytical Services
It is our standard practice to purify proteins using affinity tags (6xHis, GST, etc) but we also can purify new protein targets. Purification can involve primary purification/concentration with tangential flow filtration and one or more steps of ion exchange, hydrophobic interacitve chromatography, affinity, and/or, size-exclusion chromatography. The quality of the protein prep is evaluated by SDS PAGE. We can carry out protein purification optimisation studies if requested by the customer.
A typical purification run can take between 2-5 weeks.
The Core has the capability to offer services and analytical tools to study biomolecular interactions in the discovery and analysis of binding partners, thermodynamics and kinetic paramaters. Some common parameters we can obtain are Gibbs free energy and entropy and enthalphy changes, binding affinity, dissociation rate constants, the affinity equilibrium constant, and binding stiochiometry. Equipment to obtain these parameters are an Isothermal Titration Calorimetry (ITC) instrument and a Surface Plasmon Resonance (SPR) instrument capable of high throughput realtime analysis.
The Core has a variety of capabilities from protein production to analysis. The resources enabling these capabilities range from bioreactors/fermenters to instruments that can aid in the analysis the bio-molecular interactions. Below are descriptions of some of the equipment utilized at the Core.
Eppendorf/New Brunswick Scientific BioFlo115
A 7.5L stirred tank bioreactor system for batch, fed-batch, and spin filter perfusion culture can be utilized for scaled-up mammalian and insect cell cultures seeded from shake flask cultures.
Wave Bioreactors™ for mammalian and insect cell culture
The core has range of disposable GE Healthcare Wave Bioreactor™ systems ranging from 500ml to 25 liters in culture volume. These bioreactors are also used in pilot scale biopharmaceutical productions and facilitate protein expression from highly shear, pH, temperature and oxygen sensitive cell lines via accurate control of these parameters enabling proper mass transfer conditions for optimum growth and protein expression. Our systems are most commonly used for transient expression via the secretory pathway although this can vary depending on the target protein. Scale up is achieved from shaking or static CO2 incubator cultures. Realistic protein expression yields for transient mammalian cell expression ranges from 0.01-20 mg/L. The Baculovirus expression system yields are higher at 0.1 to 150 mg/l on average considering a similar development timeline to produce the baculovirus and infect the insect cells for protein production.
New Brunswick Scientific BioFlo610
Microbial fermentations are carried out in shake flasks cultures but can be scaled up to 40L volumes in a 65L fermenter in order to produce large batches, if required.
The system used at the Protein Production Core for large pilot scale microbial cultures is the New Brunswick Scientific BioFlo610 65L SIP fermentor / bioreactor. This industrial grade reactor is specifically designed for microbial bioprocess manufacturing.
GE Healthcare Quixstand TFF and Thermo Fisher TZ28/GK Continuous Centrifuge
For the primary recovery and concentration of cell culture product and/or cells the Core has several options with tha ability to process small and large scale volumes.
For the larger scale, a tangential flow filtration unit from GE Healthcare is capable handling 10-20L of crude cell stock while the continuous centrifuge can process 30-50L of suspended cell pellets if required.
AKTAxpress Fast Protein Liquid Chromatography (FPLC) System
The purification of proteins is greatly enhanced with two GE Healthcare AKTAxpress FPLC’s. The protein core does a variety of protein purifications including multi-dimensional chromatography to enhance the purification process of complex mixtures. Our standard protocols usually have an affinity purification followed by a desalting step and ion exchange and/or a gel filtration step for size exclusion chromatography.
Bio-Rad ProteOn XPR36 Surface Plasmon Plasmon Resonance
The Core has the capability to perform real-time bio-molecular interaction studies in a high throughput 36 reaction array format using the ProteON XPR36 instrument. This instrument allows for binding partner determination and kinetics and affinity parameters of binding to be found apart from concentration assays and thermodynamics analysis.
Isothermal Titration Calorimetry (ITC) using the GE Healthcare AutoITC200
Automated high throughput analysis of thermodynamics of binding can also be accomplished with the AutoITC200 instrument. This instrument mainly allows for the analysis of bio-molecular interactions via measure the binding affinity (Ka), enthalpy changes (ΔH), and binding stoichiometry (n) of the interaction between two or more molecules in solutions.
For a Price List, please call (407)745-2144 or email us.