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 Post Doctoral Fellow |
 Jared Schooley, rPPC Research Technologist |
OUR SERVICES
Protein Expression in Insect Cells
Protein Expression in Yeast 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 E.coli cells will be 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 and glycerol stock is prepared from grown colonies. The price applies per one vector.
Recombinant protein expression involves the process of cultivating transformed E.coli cells, so that they transcribe and translate the desired protein. The rate includes media preparation, growing overnight culture (5-10ml), inoculation into scaled up (1-10L) culture, induction, and harvest. For proteins with low- to zero-expression, and upon customer request, the fermentation will be performed using BIOSTAT Bioreactors at additional cost for Bioreactor usage. Scaled-up culture will be harvested for subsequent purification.
Mammalian rProtein expression involves 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. The cells are then either lysed to extract the expressed protein during downstream processing, or the growth medium is collected for secreted protein. The cost unit is per 1L of culture.
This service includes small (5ml) scale expression of rProtein in E. coli and mammalian cells (CHO, HEK293). It includes fermentation, induction and SDS-PAGE and/or Western Blot analysis. To approximate the production level of rProtein, a purification will be performed upon customer request at additional cost. This step precedes production scale upon customer request.
Competent DH10 E. coli are transformed with baculovirus transfer plasmid to generate recombinant baculovirus carrying the DNA of interest. PCR is performed to confirm true recombination through selection on Bluo Gal plates (Step 1; $885). Insect cells (Sf9) are then infected with recombinant baculovirus to grow and amplify virus in higher titer (two or three passages). The virus titer is determined by plaque assay (Step 2; $1300). During the final step, Sf9 cells are grown in high density and infected with amplified virus. Infected Sf9 cells are cultivated and collected in 48-72 hours, followed by lysis to recover rProtein (Step 3; $1365/1L culture). Charges applied depend on requested protocol steps.
Eukaryotic cells (mammalian/insect) to be rescued will be thawed, recultured with daily viability confirmation before freezing for long-term storage. Hybridoma will be tested for activity in ELISA for additional charge.
A number of downstream processes are used to recover active recombinant protein from bacterial or mammalian cell culture: cell separation by centrifugation, cell lysis, and membrane disruption by high pressure homogenizer. Subsequent lysate is clarified using high speed centrifugation. The unit cost is per cell mass from 1L of culture.
This service includes purification of rProteins from cell lysate or culture medium via special affinity label (tag – 6xHis, HA, cMYC; or fusion protein – GST, MBP). The purification is performed by AKTA chromatography system. The charge applies to one step of affinity purification of 6xHis-tagged protein, plus cost of affinity resin for GST, MBP, HA-, cMyc- or FLAG-tagged proteins. Any additional step (Size Exclusion, Ion Exchange) is charged separately and includes labor time and instrument usage cost.
TEV protease is used to remove affinity tags from purified recombinant fusion proteins. The rProtein will be incubated with TEV protease followed by separation of TEV protease and tagless rProtein on affinity column using AKTAxpress FPLC system. Quality control is performed by SDS-PAGE analysis.
This service produces a large amount (mg quantities) of antibody in stationary bioreactor. Hybridoma cells are grown in high density and culture medium (1L) is collected for subsequent antibody purification. *If the cells grown in stationary type of bioreactor, the price applies to 50 mL unit of highly concentrated hybridoma supernatant.
Antibodies are purified via automatic liquid chromatography system. The technique separates antibody on the basis of a reversible interaction between the antibody and a specific ligand coupled to a chromatography matrix (protein G, protein A). Price is per 1L. If the highly concentrated hybridoma supernatant (from bioreactor) is to be purified, the above price applies to 50 mL unit.
The DNA provided by customer will be expressed in competent E.coli cells. Transformed E. coli are cultivated and DNA will be extracted and purified using plasmid mini- or maxi-prep kit, depending on customer’s request. Quality control of DNA prep includes appearance on agarose gel, A260/280 ratio, concentration, and quantitation by Nanodrop. Cost depends on DNA quantity requested.
The samples from analytical or production scale of rProtein expression will be analysed by SDS-PAGE. The charge applies per 1 mini-gel/10 samples.
This services 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, 10 wells], includes labor time and consumables). Upon customer request, the assay can be performed using fully automated Wes Simple Western system from ProteinSimple. In such a case, the rate will include the instrument rental time (minimum 3 hours).
rPPC offers instrument time sharing on daily rates (Bioreactors – Qplus 1Lx3, Bplus 5, High Pressure Homogenizer, AKTAxpress, Profinia FPLC, YSI-Bioanalyzer, 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 assistance in instrument set up, calibration, washing and turning off.
Consultation time provided by Dr. Izolda Popova on any issue related to the services provided by rPPC is considered free of charge for the first 15 minutes. For longer consultations, the rates for Technical/Instrument Assistance Time apply.
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) |
$65.00
|
$65.00
|
$104.00
|
$162.50
|
| Protein expression in E.Coli or Yeast (per 1L) |
$352.00
|
$352.00
|
$563.20
|
$880.00
|
Protein expression in mammalian cell system
(per 1L) |
$3306.00
|
$3306.00
|
$5289.60
|
$8265.00
|
Analytical (small-scale) Expression
(per 100mL) |
$365.00
|
$365.00
|
$584.00
|
$912.50
|
Protein expression in insect cell system
(per 1L) |
$3657.00
|
$3657.00
|
$5851.20
|
$9142.50
|
Protein Downstream Processing
(per cell lysate from 1L) |
$363.00
|
$363.00
|
$580.80
|
$907.50
|
Recombinant Protein Purification
(per 1L) |
$209.00
|
$209.00
|
$334.40
|
$522.50
|
Tag Cleavage with TEV Protease
(per 1L) |
$239.00
|
$239.00
|
$382.40
|
$597.50
|
Large Scale mAb Production
(concentrated per 50ml or Non-Concentrated per 1L) |
$815.00
|
$815.00
|
$1304.00
|
$2037.50
|
mAb Purification
(per 50 ml Concentrated or per 1 L non-concentrated) |
$815.00
|
$815.00
|
$1304.00
|
$2037.50
|
Rescuing and Cultivating Eukaryotic Cells
(per 1 cell line) |
$330.00
|
$330.00
|
$528.00
|
$825.00
|
Plasmid Propagation & Purification
(per plasmid) |
$268.00
|
$268.00
|
$428.80
|
$670.00
|
| Instrument time – daily rate (Bioreactor Qplus 1Lx3, Bplus 5L, Homogenizer, AKTAxpress, Profinia FPLC, YSI Bioanalyzer, Innova Incubator Shaker) |
$51.00
|
$51.00
|
$81.60
|
$127.50
|
| 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
|
SDS-PAGE Analysis
(per 9 samples) |
$135.00
|
$135.00
|
$216.00
|
$337.50
|
Western Blot Analysis
(per 9 samples) |
$185.00
|
$185.00
|
$296.00
|
$462.50
|
