ABOUT US
Welcome to the Recombinant Protein Production Core!
The Recombinant Protein Production Core (rPPC) provides quality controlled recombinant proteins for researchers within the Northwestern Community (WCAS, McCormick, Feinberg) and also serves academic and industry researchers outside of Northwestern University. rPPC operates based on the two service models: (1) a Training model where Northwestern researchers use specialized bioreactor systems and participate in hands-on-training activities and (2) a Production model where staff carry out expression (mg to gm scale) and purification of recombinant or synthetic biologics, including potential therapeutic proteins and peptides, among others. Currently, the main focus of rPPC is to be a user-facility; the facility has parallel bioreactor systems for multiplexed lab-scale cultivation of microbial, insect, and mammalian cells. rPPC also serves as a production facility, providing low-cost recombinant biologics for researchers at Northwestern University.
rPPC’s goal is to transform research at Northwestern University by addressing a variety of unmet needs that can often stall cutting-edge projects. As a centralized protein production center rPPC facilitates the expression of proteins for a broad spectrum of research activities ranging from structure/function studies to producing protein therapeutic candidates such as monoclonal antibodies for in vitro and in vivo pre-clinical studies. Scalable, high-throughput cultivation enables screening mutants for phenotype studies, proteomics, stem cell characterization, media screening, strain and process optimization for the production of biochemicals, e.g. biofuels. High-throughput cell-free protein production interfaces well with HTA to “augment and expand Northwestern University’s community of multidisciplinary researchers focusing on areas of biomedical research relevant to NIH.” Protein purification completes the facility enabling a one-stop shop for researchers to clone, overexpress, and purify relevant biologics.
The rPPC is co-directed by Michael Jewett, PhD, and Keith EJ Tyo, PhD. The co-directors bring top level expertise to implementing, building, and overseeing the direction of the institute; they are also involved in initial project planning and ensuring that projects adhere to agreed upon timelines and that deliverable deadlines are met. The day-to-day scientific operations of the rPPC are managed by Izolda Popova, MD, PhD. Dr. Popova also advises on selection and design of optimal overexpression systems, optimizing protein expression, and provides hands-on-training for cell cultivation. Dr. Pshenychnyi is responsible for technical execution of research projects.
 Dr. Keith Tyo, rPPC Co-Director |
 Dr. Michael Jewett, rPPC Co-Director |
 Dr. Izolda Popova, rPPC Assistant Director |
 Dr. Sergii Pshenychnyi, rPPC Research Associate |
 Jared Schooley, rPPC Research Technologist |
OUR SERVICES
Protein Expression in Insect Cells
Protein Expression in Mammalian Cells
Protein Expression in Bacterial Cells
rPPC offers its services based on two service models: Training model and Production model. In accordance with the Training model, Northwestern researchers can use the rPPC specialized bioreactor systems and partake in hands-on-training activities. Consistent with the Production model the rPPC staff performs expression (mg to gm scale) and purification of recombinant or synthetic biologics including potential therapeutic proteins and peptides, among others. Please see below for a full list of our services:
Electrocompetent or Chemically Competent E.coli cells are transformed by electroporation or heat shock with an expression vector carrying the target DNA, encoding the protein of interest.Transformed cells are plated to selection agar plates, glycerol stock is prepared from grown colonies.
Mammalian and Insect cells are transfected with target DNA encoding the protein of interest.
The service includes small (5mL) scale expression of rProtein in E.coli and mammalian cells (CHO, HEK 293). It includes fermentation, induction and SDS-PAGE analysis.
Recombinant protein expression involves the process of cultivation of transformed E.coli cells in 1L shaker flasks, 1L fermenter (up to 9 in parallel) or 5L fermenter. An additional rate is applied to expression using Q-plus and B-plus Bioreactors due to routine daily maintenance and cost of consumables (reagents, tubing, oxygen, syringes, air filters).
rProtein expression involve inserting a DNA vector, encoding protein of interest, into mammalian cells (CHO, HEK293) and then culturing cells so that they transcribe and translate the desired protein.
Insect cell cultures are transfected with wild type baculovirus and baculovirus transfer plasmid to generate recombinant baculovirus carrying a DNA of interest. Competent DH10 E.coli are transformed with baculovirus transfer plasmid to generate recombinant baculovirus carrying a DNA of interest. A PCR is performed to confirm true recombination through selection on Bluo Gal plates (Step1). Insect cells then infected with recombinant baculovirus to grow and amplify virus in higher titer (two ot three passages). The virus titer is determined by plaque assay (Step 2). During final step the cells are grown in high density and infected with amplified virus. Infected cells are cultivated and collected in 48-72 hours, followed by lysis to recover rProtein (Step 3). Charges applied depending on requested protocol steps and according to customers’ price group.
Generation of hybridoma (secreting antibody) includes 2 steps: 1) Fusion of immune splenocytes and myeloma to make hybrid cells; 2) screening of newly formed hybridomas in ELISA. After testing the plates are transferred to the customer for in-lab cultivation and preservation of desirable clones.
An eukaryotic cells (mammalian, insect, hybridoma) to be rescued will be thawed, re-cultured with daily viability checking before freezing down for long time storage. Hybridoma will be tested for activity in ELISA for additional charge.
The downstream processing involves cell mass separation by centrifugation, cells membrane disruption and lysis by high pressure homogenizer, followed by lysate clarification using high speed centrifugation. The unit cost is per cell mass from 1L of culture.
The service includes purification of rProteins from the cell lysate or culture medium using AKTAxpress chromatography system. The charge applies to one step of purification per 1L of cell culture.
TEV protease is used to remove affinity tags from purified recombinant fusion proteins. The rProtein is incubated with TEV protease followed by separation of TEV protease and tag-less rProtein on affinity column using AKTAxpress FPLC system.
Hybridoma cells are grown in high density and culture medium (1L) is collected for subsequent antibody purification. *If the cells are grown in stationary type of bioreactor, the price applies to 50 mL unit of highly concentrated hybridoma supernatant.
The competent E.coli cells will be transformed with the template DNA provided by customer. Transformed E.coli are cultivated and DNA mini or maxi prep are extracted. Quality control of DNA prep includes appearance on agarose gel, A260/280 ratio, concentration, quantitation by NanoDrop.
The samples from analytical or production scale of rProtein expression will be analysed by SDS-PAGE. The charge applies per 1 mini-gel.
The service includes an analytical immune detection of a protein in the sample or cell lysates produced by rPPC upon customer request, or in the sample provided by a customer. The assay can be performed using traditional gel-blot-membrane protocol (the cost for traditional Western Blot (1 precast gel) includes labor time and consumables) or, upon customer request, by fully automated Wes Simple Western from ProteinSimple. In such a case the rate will include additionally instrument renting time (minimum 3 hours).
rPPC offers instrument time sharing on daily rates (Bioreactors – Qplus 1Lx3, Bplus 5L, High Pressure Homogenizer, AKTAxpress, Profinia FPLC, Innova-44 Incubator Shaker) and hourly rates (High Speed Centrifuge, Optima L-90 Ultracentrifuge, Synergy H1M Microplate Reader, Bio-Rad Gene Pulser XCell Electroporator, Bio-Rad T100 Thermal Cycler, NanoDrop 2000 Spectrophotometer, The Wes Simple Western Assay) based on instrument category. rPPC also offers an hourly rate for assisted instrument use. This is in addition to the instrument use rates. It also includes instrument training time.
Technical assistance includes steps in research protocols not included in listed rPPC services (mixing the samples, aliquoting the samples, heat in oven, mixing the solutions). It also includes instrument training time, assistance in instrument set up, calibration, washing and turning off.
The service includes protein solubilization by denaturing agents followed by protein refolding using dialysis method. The rate applies per 1g of wet inclusion bodies pellet.
SERVICE FEES
The rPPC follows a mixed-use model and has a fee structure that bills for instrument time, specific services (i.e. analytical method development), or nuts to bolts projects. The latter scenario permits an investigator to drop off a cDNA and later receive: a) a cell line expressing recombinant protein, b) a data package describing the cell line (i.e. specific expression in pcd), purification, characterization (purity, potency, etc.), and/or c) a vial of purified protein in the amount requested for the project.
The fees for all of offered rPPC services are listed as follows:
|
Service
|
Fees
|
|
NU users
|
External users, CBC Open Access
|
External users, Academic
|
External users, Corporate
|
| Transfection/Transformation (per one vector) |
$172.00
|
$172.00
|
$275.20
|
$430.00
|
| Analytical (Small-scale) expression in E.coli and mammalian cells |
$437.00
|
$437.00
|
$699.20
|
$1,092.50
|
| Recombinant Protein (rProtein) expression in E.coli/Shaker Flasks (per 1L of culture) |
$377.00
|
$377.00
|
$603.20
|
$942.50
|
| Recombinant Protein (rProtein) expression in E.coli/Bioreactor Bplus (per 1L of culture) |
$568.00
|
$568.00
|
$908.80
|
$1,420.00
|
| rProtein expression in mammalian cell system (per 1L of culture) |
$3,180.00
|
$3,180.00
|
$5,088
|
$7,950
|
| Protein expression in insect cell system (per 1L of culture) |
$3657.00
|
$3657.00
|
$5851.20
|
$9142.50
|
| Generation of Mouse Hybridoma, Producing Monoclonal Antibodies (per one fusion) |
$3,160.00
|
$3,160.00
|
$5,056.00
|
$7,900.00
|
| Large Scale mAb Production, Concentrated per 50ml or Non-Concentrated (per 1L of culture) |
$815.00
|
$815.00
|
$1,304
|
$2,037.50
|
| Downstream processing of grown culture (before rProtein purification) (per cell mass from 1L culture) |
$363.00
|
$363.00
|
$580.80
|
$907.50
|
| Recombinant Protein Purification (per lysate from 1L culture) |
$312.00
|
$312.00
|
$499.20
|
$780.00
|
Tag Cleavage with TEV Protease
(per protein) |
$514.00
|
$514.00
|
$822.40
|
$1,285.00
|
DNA Plasmid Propagation & Purification
(per plasmid) |
$302.00
|
$302.00
|
$483.20
|
$755.00
|
Rescuing and Cultivating Eukaryotic Cells
(per 1 cell line) |
$330.00
|
$330.00
|
$528.00
|
$825.00
|
Protein recovering from inclusion bodies
(per 1 protein) |
$357.00
|
$357.00
|
$571.20
|
$892.50
|
| SDS-PAGE Analysis |
$161.00
|
$161.00
|
$257.60
|
$402.50
|
| Western Blot Analysis |
$268.00
|
$268.00
|
$428.80
|
$670.00
|
| Instrument time – daily rate 1 (Innova Incubator Shaker) |
$51.00
|
$51.00
|
$81.60
|
$127.50
|
| Instrument time – daily rate 2 ( Profinia FPLC, High Pressure Homogenizer, Orbital shaker in CO2 incubator) |
$65.00
|
$65.00
|
$104.00
|
$162.50
|
| Instrument time – daily rate 3 (Q and Bplus Bioreactors, AKTAxpress FPLC) |
$100.00
|
$100.00
|
$160.00
|
$250.00
|
| Instrument time – hourly rate (High Speed Centrifuge, Ultracentrifuge, Microplate Reader, Gel Imager, NanoDrop Spectrophotometer, Gene Pulser, Thermal Cycler, The Wes Simple Western) |
$19.00
|
$19.00
|
$30.40
|
$47.50
|
| Technical/Instrument Assistance time – hourly rate |
$83.00
|
$83.00
|
$132.80
|
$207.50
|
