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University of Colorado Cancer Center

University of Colorado Cancer Center, A National Cancer Institute-designated Comprehensive Cancer Center
Tissue Culture Shared Resource at CU Cancer Center
 

Protein Production/MoAB/Tissue Culture


The Protein Production, Monoclonal Antibody, Tissue Culture Shared Resource (PPSR) supports
basic and translational research at CU Cancer Center Members with validated cell lines, preparation of hybridomas, production of monoclonal antibodies, isolation and titering of baculovirus expressing recombinant proteins, and large scale preparation of recombinant proteins and monoclonal antibodies.
 
PPSR provides centralized services to produce cancer-related proteins for CU Cancer Center members to study their mechanism of action in identified drugs and cancer-related biomarkers.
 

Services:

Protein Services

 
Mammalian System
Mammalian cells are the preferred expression platform for proteins requiring fully mammalian post-translational modifications. As such, they are commonly used for the production of antibodies and therapeutic proteins. They are also preferred when the downstream application for a recombinant protein is in a functional mammalian cell-based assay
 
Phase I: Gene Synthesis and Subcloning – performed by Research Lab
Customer produces cDNA of any desired coding sequence and subclone it into a mammalian expression vector. Once cloned, all constructs are restriction digest and sequence verified.  Customer provides required amount of DNA for size of transfection (general-3 μg DNA/ml transfection vol).
 
Phase II: Pilot Protein Expression
Generally, we conduct small scale expression  trials to identify the best clones and/or expression conditions. We use these conditions to continue on the Phase III.
 
Phase III: Protein Expression
For larger protein requests, we scale up expression using optimized conditions. We utilize 2-10L Wave Bag systems to produce gram quantities of the desired protein.
 
Baculovirus System

The baculovirus expression vector system (BEVS) is a powerful system for the production of high quality proteins. Although time lines are increased compared to E. coli, proteins produced in BEVS tend to be soluble and functional and carry post-translational modifications like phosphorylation and glycosylation. The system gives very high recombinant protein yields and we often recommend it for proteins that are difficult to express in E. coli.

Phase I: Gene Synthesis, Subcloning, and Baculovirus Paration
Customer provides cDNA or optimize with any desired coding sequence and subclones it into a baculovirus transfer vector. Once cloned, all constructs are restriction digest and sequence verified. Transfer vectors are given to us and are used to generate recombinant bacmids and P1, P2, and P3 virus stocks.

Phase II: Protein Expression and Purification
We infect insect cells with high titer P3 viral stock and determine optimal conditions for protein expression and purification.

Phase III: Larger Scale Protein Expression and Purification
Should additional quantities of protein be required we scale up expression by ustilizing 2-10L Wave /bag system.

Monoclonal Antibody Services

 
Custom Hybridoma Services/Monoclonal Antibody Production


Cell Line Repository/General Cell Culture Services

  • Seeds cultures/frozen ampules
  • >200 authenticated cancer cell lines, including 75 different lung cancer cell lines and 26 breast cancer cell lines 
  • Large scale cell culture production
  • List of human cell lines available
  • Mycoplasma testing (same day results) 
  • Cell Line Authentication with Certificate of Authentication for publication requirements

Hyclone On-Site FBS/Media Supply Center

  • Hyclone characterized FBS tested/reserved Lot-freezer located in rm5404B from 8-3, distributed by shared resource staff
  • Hyclone Prices/order form
  • Hyclone Cell Culture Media and Supplements located in the hall RC1-S 5th floor hall.