Protein Expression and Purification Services
The UPenn HTSC offers protein expression services in E. coli, insect cells, and mammalian cells. We encourage users to consult with the Technical Director regarding the best design for a protein expression and purification project.
Expression Plasmid Design
Core staff will advise users on the design of expression constructs to meet the specific needs of each research project. An organism specific codon optimized expression vector will be ordered and prepared.
Bacterial Expression systems
We offer scaled protein expression services in E. coli. The advantage of bacterial systems for protein expression include: the economics, diversity of vectors available for expression of tagged proteins, and availability of E. coli strains for expression. We accept most expression vector backbones for these services.
- Expression and Solubility Study
The integrity, stability, solubility, and yield of proteins will be evaluated in different bacterial strains and induction conditions (e.g., temperature, induction time) and assessed by Coomassie blue stained SDS-PAGE and small scale one-step affinity purifications
- Large-Scale Production of Recombinant Proteins
Recombinant proteins will be expressed using optimized conditions for expression of soluble protein in 1L shake cultures.
Baculovirus Expression System
Insect cells are a scalable expression system amenable for proteins that are difficult to express in E. coli, including large proteins and multisubunit protein complexes. For derivation of new baculoviruses, we accept all transfer vector plasmid backbones (e.g. pFastBac, pACEBac, pLIB/pBIG, BacMam) that contain the Tn7L and Tn7R recombination sites, the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) transposition method.
- Bacmid Preparation
A recombinant bacmid DNA is prepared by transformation of transfer vector plasmid DNA into E. coli DH10EMBacY cells. The source of genomic baculovirus DNA is from Autographica californica multicapsid nuclear polyhedrosis virus (AcMNPV) and the DH10EMBacY genome contains deletions of the v-cath cysteine protease and the chiA chitinase genes, which significantly reduces the viral proteolytic activity and the cell lysis. In addition, the DH10EMBacY genome contains the YFP gene under the control of the polH promoter, which enables virus performance and protein production to be monitored by measuring the YFP fluorescence. Successful transposition of the GOI into the bacmid disrupts the lacZa gene, which enables blue/white selection on agar plates containing X-gal to identify positive clones. Recombinant bacmid DNA is isolated from positive (white) colonies and recombination further confirmed by PCR.
- Bacmid DNA Transfection to prepare high-titer baculovirus
A suspension culture of Spodoptera frugiperda (Sf9) insect cells cultured in SFM are transfected with Cellfectin (Invitrogen) and a PCR-verified recombinant bacmid DNA. The tissue culture supernatant from transfected cells is harvested 120 hours post-transfection and considered the P1 virus stock. Typical virus titers of P1 stocks with our methods are in the 1-2 x 108 pfu/ml range.
- Amplification of High-titer Recombinant Baculovirus Stocks
High titer P2 virus stock is produced from the P1 virus (the original virus from cell culture supernatant of transfected cells) in Sf9 cells grown in SFM as a suspension culture. A high-titer P2/P3 virus stock is generally produced by infecting Sf9 cells with a virus stock at a low multiplicity of infection (MOI=0.1) and culturing for 120 hours. Typical viral titers are in the 1-2 x 108 pfu/ml range.
- Baculovirus tittering
Viral titers of newly amplified stocks can be determined by automated microscopy of infected Sf9 cells stained with anti-GP64 antibodies.
- Time-Course Study of Protein Expression
A 100 ml suspension culture of Sf9 cells grown in SFM is infected with a high-titer baculovirus stock at an MOI=1-2. Cells or conditioned media (for secreted proteins) are harvested at 24, 48, and 72 hours post infection to evaluate the integrity, stability, solubility, and optimum yield of the desired protein(s) by Western Blotting or pilot small-scale purifications.
- Large-Scale Production of Recombinant Proteins
A high-density (1 x106 cells/ml) suspension culture of Sf9 cells grown in SFM is infected with a high-titer virus at a multiplicity of infection (MOI) of 1-2 for 48 to 72 hours in shake flasks. Users are provided a cell pellet (or supernatant for secreted proteins). We routinely infect cultures of 250 ml upto 1 L of insect cells.
Mammalian Expression System
We offer a service for the transient expression of proteins in high-density Expi293 suspension cultures using standard plasmid transfection protocols with a transcription boost post-transfection. We offer services to transfect cultures of 100 ml upto 1 L of cells in shake flasks.
- Time-Course Study of Protein Expression
A 100 ml culture of Expi293 cells is transfected with a mammalian expression plasmid. Cells and/or conditioned media (for secreted proteins) are harvested at 24, 48, 72, 96 hours post-boost to evaluate the integrity, stability, solubility, and optimum yield of the desired protein(s) by Western Blotting or pilot small-scale purifications.
- Large-Scale Production of Recombinant Proteins
A high-density (1 x106 cells/ml) suspension culture (100ml, 250ml or 500ml) of Expi293 cells in shake flasks is transfected with a mammalian expression plasmid. Users are provided a cell pellet (or supernatant for secreted proteins) at a time point when expression and stability of the protein was pre-determined to be optimal. Our infrastructure is capable of multiple 500ml shake cultures to achieve a desired yield.
Protein Purification
We offer purification services for proteins expressed in E. coli, insect cells, or mammalian cells. Purification services will include cell lysis, purification using a combination of affinity (e.g. Ni2+-chelate, GSH-, etc.) and conventional (e.g. ion exchange, size exclusion) chromatography steps. Purified proteins will be buffer exchanged, concentrated, and assessed by SDS-PAGE analysis. We encourage users to consult with the Technical Director regarding the best design for a purification project. For purification services from other host source systems (e.g. hybridomas), please contact the technical director.