Protein Expression and Libraries Facility

Services

The Protein Expression and Libraries Facility is a fee-for-service center dedicated to providing technical assistance in the following areas:

• Consultation and training
• Recombinant DNA engineering
• Recombinant protein expression in bacteria
• Recombinant protein production via Baculovirus Expression Systems (BVES)
• Mammalian cell expression system
• Protein purification
• Retrovirus production
• Other

Consultation and training

Each project is initiated with consultation to choose the appropriate expression system and to ensure that the recombinant DNA plasmid is engineered to meet the needs of the investigator. When requested, the facility staff provides technical training in all aspects of services provided by the Facility.  The goal is to educate users about the different systems and what to expect from the various services provided.  The Facility also sponsors informational seminars about cloning technologies, expression vector technologies, genomic library resources, and applications for
genomic libraries.

Recombinant DNA engineering

The facility provides consultation and design of a cloning strategy (i.e., conventional, TOPO™, Gateway™, and Quickchange site directed mutagenesis) appropriate to meet the goals of the project.  The nucleic acids of interest (e.g., cDNA, promoter, etc) are isolated from a customer-supplied vector and subcloned into a new vector.   Newly derived plasmid DNAs are authenticated by restriction endonuclease mapping and DNA sequencing, and amplified/prepared for transfection based applications.

*Note: Customers will be responsible for the costs incurred in the subcloning service if the sequence of the supplied plasmid does not correspond with the sequence provided.

For a list of available vectors, click here

Recombinant protein production in bacteria

Use of prokaryotic expression systems offers an economical method to achieve production of large amounts of recombinant protein.  The Facility provides technical support for production of recombinant protein by regulated, high-level intracellular expression in bacteria. The core maintains a repository of inducible bacterial expression vector technologies, including pET (Stratagene), pGEX (GE Healthcare), pRSET (Invitrogen), pQE30 (Qiagen), pDUET (Novagen).  Click here for a complete list of bacterial expression vectors.  This collection of vectors will allow for fusion of epitope tags (alone or in combination) to the protein of interest (GOI), including GST (Glutathione-S-transferase), 6 histidines (6His), SUMO, FLAG, or Streptag II at the NH3- or COOH-terminus of a protein.  High level protein expression (analytical or preparative scale) is achieved by IPTG induction of T7/T5 RNA polymerases in high-performance Epicuran Coli â BL21(DE3), BL21-Gold (DE3), C41(DE3), BL21(DE3)-pRIL, Origami (DE3), Rosetta (DE3), Rosetta-gami (DE3), B834 (DE3), and PL21-AIä competent cells (Stratagen, Novagen, Invitrogen). 

Available services

• Optimization of soluble protein expression
• Preparative production

Optimization of soluble protein expression

The purpose of this service is to optimize the expression of soluble protein. Because some proteins fold poorly or incompletely when over-expressed in E. coli, the Facility uses several strategies to adjust variables that account for inclusion body formation such as N-terminal protein fusion to soluble tags (e.g. GST, SUMO), culture medium and aeration, growth temperature (18°C-37°C), expression vector characteristics including promoter activity, and use of bacterial strains engineered to express underrepresented tRNA species during production.

Bacterial expression vectors containing a gene of interest are transformed into a strain of bacteria (e.g. BL21) optimized for recombinant protein expression.  An analytical scale (100 ml) induction of protein expression is used to determine the extent of the recombinant protein’s solubility under the defined experimental conditions by SDS-PAGE and Coomassie stain

Preparative production

The laboratory has the capacity to do up to 12 L per a day of large-scale recombinant protein production in bacteria.

Recombinant protein production via baculovirus expression systems

Baculovirus expression systems (BVES) represent an alternative approach to produce large amounts of properly folded and functional recombinant proteins. The benefits of protein expression with baculovirus include the virus being able to accommodate large inserts, eukaryotic post-translational modification, enhanced protein folding and function, high expression levels, easy scale up with high-density suspension culture, and safety.  The Facility supports generation of baculovirus vectors from nearly all commercially available BVES.  Click here to view a list of available baculovirus expression vectors.  Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors, have facility staff engineer the plasmid DNA construct, or to provide the facility with small volumes of previously prepared virus stock.

Available services:

• Generation of High-titer baculovirus stock
• Amplification of high-titer baculovirus stocks
• Analytical scale productions for optimization of protein expression
• Preparative scale productions

Generation of high-titer baculovirus stock

The generation of recombinant baculovirus is achieved via one of two approaches. Spodoptera frugiperda (Sf9) insect cells are cotransfected with the transfer vector plasmid DNA containing the foreign gene to be expressed and baculovirus DNA from Autographica californica nuclear polyhedrosis virus (AcNPV)-Baculogold, Bac-N-Blue, and BacPak methods.  Alternatively, insect cells are transfected with a recombinant bacmid DNA recovered by transposition of the transfer vector DNA in E. coli cells (DH10Bac), the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) method.  Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors or have facility staff engineer the plasmid DNA construct.  For more information about baculovirus expression systems click here.

Baculogold (Pharmingen, Sigma)-3 weeks

• Transfection of insect cells (Sf9) with the recombinant transfer vector (containing gene of interest to be expressed) and baculoviral DNA.
• Amplification of high-titer recombinant baculovirus stock (100 ml-P2).
• (Optional) virus tittering.

Bac-to-Bac (Invitrogen)-2 weeks

• Transposition of pFastBac transfer vector in DH10Bac, Bacmid preparation, PCR comfirmation of bacmid transposition.
• Transfection of insect cells (Sf9) with Bacmid DNA.
• Amplification of high-titer recombinant baculovirus stock (100 ml-P2).
• (Optional) virus tittering.

Bac-N-Blu/BacPak (Invitrogen/Clontech)-6 weeks

• Transfection of insect cells (Sf9) with the recombinant transfer vector (containing gene of interest to be expressed) and baculoviral DNA.
• One round of plaque purification/virus amplification.
• Amplification of high-titer recombinant baculovirus stock (100 ml-P2).
• (Optional) virus tittering.

The BaculoGold™ and Bac-to-Bac™ methods are designed to achieve virtually 100% recombination efficiencies and recombinant protein expression is subsequently evaluated using recombinant virus amplified (without plaque purification) in P2 in insect cells.  A single round of plaque purification of recombinant virus from the initial virus production (P1 virus) is optional before virus is amplified in a second passage (P2) for these methods.  Recombinant baculovirus derived from all other commercially available baculovirus DNA preparations is produced with 80-90% efficiency and requires plaque purification to remove parental virus.

Analytical scale productions for optimization of protein expression

 The purpose of this service is to aid investigators in assessing the optimal conditions for protein production. The results can be used to determine optimal conditions for protein production. There are numerous examples where the integrity, stability and biological activity of recombinant proteins vary with time after infection.  Therefore the recommendation of the facility is that each new baculovirus for a protein be extensively characterized in analytical scale productions before proceeding to large-scale productions.

This service entails the infection of a 100 ml suspension culture of upto three insect cell lines [Sf21, Sf9, and High Five (T.ni)] using a high-titer baculovirus stock.  Cells or conditioned media (for secreted proteins) are harvested at 24, 48, and 72 hours post infection. Harvested samples are analyzed for expression of the recombinant protein by western blot.   Investigators can complete the Western blot analysis or have the facility staff complete the analysis for an additional fee (customer must provide appropriate gene specific antibodies, if desired).

Amplification of high-titer baculovirus stocks

The purpose of this service is to prepare high-titer (>108 pfu/ml) baculovirus stocks in serum free medium for preparative scale productions of recombinant protein in insect cells. Titration of the final high-titer stock is an optional service.

Preparative scale productions

Expression of recombinant protein under optimized conditions in high-density (1-2 x106 cells/ml) suspension cultures (50 ml-1L) of Sf9, Sf21, or Trichoplusia ni (commonly referred to as High Five) cells at a multiplicity of infection (MOI) equal to 1-2.  We recommend use of High Five cells for production of secreted proteins.

1 We do not guarantee the yield of purified protein/volume of culture since it changes from protein to protein. To achieve a certain yield, additional culture volumes may be required.

• Multi-liter discounting is available.

Mammalian cell expression systems  

The Facility offers support for constitutive and conditional expression of cDNAs and shRNAs in mammalian cells. The Facility maintains a repository of mammalian expression vectors that possess diverse promoters for high-level constitutive or tetracycline-regulated expression (e.g., CMV, EF1a, Ubiquitin C), selection markers (e.g. antibiotic resistance, fluorescent protein, cell surface markers) for establishment of stable cell lines, and epitope tags (e.g., FLAG, HA, 6-His, GFP) for protein detection and purification.   This repository is composed of vectors (pcDNA3 based, Invitrogen) for traditional transfection-based delivery of cDNAs and newly developed retroviral delivery systems to facilitate production of high-titer viruses for gene delivery to difficult-to-transfect cell lines, primary cell types, and cancer stem cell populations.  In addition to vector systems for delivery of cDNAs, the Facility also supports vectors for delivery and expression of shRNAs in mammalian cells.

For a list of available vectors, click here.

Recombinant protein purification

The Facility provides analytical and preparative scale, one- or two-step purification of recombinant proteins expressed in bacteria, baculovirus infected insect cells, or mammalian cell lines.  The expectation is to provide highly purified recombinant proteins for use in structure-function studies, crystallographic efforts, assay development for high-throughput small molecule screening, custom antibody productions, and peptide identification of macromolecular protein complexes. Since most recombinant protein expression in bacterial and baculovirus systems takes advantage of epitope tags for monitoring expression, proteins are purified from soluble extracts using appropriate affinity matrices (e.g., Ni2+-/Co2+-agarose, GSH-sepharose, Streptactin-agarose, anti-FLAG/HA agarose) that allow for selection of specific tags (e.g. 6His, GST).  When necessary, a second purification step (e.g., affinity, size exclusion chromatography, etc) is included to ensure maximum purity of the recombinant protein.  The core cleaves epitope tags, when requested, and the recombinant protein will be further purified from tags and proteases.  Purified proteins are dialyzed into appropriate buffers and concentrated to desired concentrations.

Vector Production

The development of viral delivery systems for mammalian cells has greatly enhanced the use of genetic based experiments to dissect the role of a particular protein or network of proteins in a specific cell type or biological process.  The goal of the vector production unit is to assist Cancer Center investigators with the production of high-titer retrovirus (e.g. lentivirus). The centralization of such services provides economy of scale, high-throughput production, increased biosafety, and quality control in the virus production process.  The Core provides a variety of services including prepackaged vector preparations, custom vector packaging, and vector titrations.

Available Services

1. Distribution of prepackaged virus

2. Custom lentivirus preparation

3. Vector concentration by ultracentrifugation

4. Vector titration

5. Policies for custom vector production

Distribution of prepackaged lentivirus vectors:

1. CMV/EF1a -GFP concentrated vector
2. CMV/EF1a - mCherry concentrated vector
   These lentivirus vectors express eGFP or mCherry under the strong constitutive CMV or EF1a promoter and are useful for marking cell populations and to experimentally determine the amount of virus required for infecting a cell line.
3. EF1a-FFluciferase/mcherry concentrated vector
   This lentivirus vector expresses firefly luciferase and mCherry  from a strong constitutive EF1a promoter and is useful for monitoring cell populations in vitro and in vivo.
4. pLKO.1-non-target shRNA control concentrated vector
   This lentivirus serves as negative control for TRC (pLKO.1) shRNA plasmids.  Note: pLKO.1 vectors do not have a fluorescent marker. These ready-to-go lentivirus preparations are not titrated, however, their titer is expected to be
   equally high as fluorescent lentivirus vectors.
5. pGIPZ-scrambled shRNA concentrated lentiviral vector.
   This lentivirus serves as negative control for pGIPZ shRNA plasmids Open Biosystems.

*All prepackaged vector preparations are VSVg pseudotyped and come as filtered, concentrated vector in PBS. Concentration is performed by ultracentrifugation.  All vectors, except pLKO-scramble, are quantitatively titrated using fluorescence activation and flow cytometry using 293T cells.

**The Wistar Institute Vector production unit has purchased the above plasmids from Sigma-Aldrich and Open Biosystems and does not profit from the sale of lentivirus in any way. All lentiviral vectors produced by the Wistar Vector Production Unit  and distributed to Cancer Center laboratories are for use in research only. The Wistar Institute is in a partnership program with both Sigma-Aldrich and Open Biosystems.

Custom packaging of vectors

The facility will prepare VSVg pseudotyped vectors by:

• Transfecting retrovirus vector, 2nd generation packaging plasmid, and VSVg plasmid into HEK293T cells
• Collect viral supernatants at 12-24 hr intervals

* All packaging plasmids will be provided by the facility.

Concentration of Vector by ultracentrifugation

The Vector Production unit will concentrate vector preparations (options C and D only) by ultracentrifugation by ~1000X.  All concentrated vectors are provided in PBS as 5-10 ul aliquots.

Vector titration

Vector production unit determines titers of vectors using a quantitative titration protocol on 293T cells, if the vector contains a fluorescent marker.  Please do NOT request titrations on other cell lines or using alternative methods.

Guidelines for custom lentivirus preparation:

1. Customer billing information and a valid grant number (Wistar investigators only) or PO# (all external users) must be provided prior to the initiation of any work.
   
   
2. Provide the appropriate amount of transfection quality (e.g. Qiagen, Wizard prep, etc.) lentiviral vector DNA, containing your gene of interest, which you want to have packaged into the virus with supporting submission form. Note: WE DO NOT ACCEPT DNA MINIPREPS.
   
   
3. Indicate the concentration of your transfer vector (we prefer a working concentration of 0.5-1μg/μL).
   
   
4. In the interest of safety, please provide information on the nature of your gene of interest and its promoter (i.e.: oncogene, etc...).
   
   
5. Indicate specifics (volume of cell supernatant, concentration, titration) related to services requested.
6. Due to variability in packaging of different retroviral vectors, we do not guarantee the production of a minimum titer.  We recommend, whenever possible, that newly produced vectors are titrated.
   
   
7. Please acknowledge the Protein Expression and Libraries Facility if viruses are used in the generation of publication data.
   
   
8. We appreciate your feedback!

Other Services

• Preparation and distribution of protein expression plasmids
• Vector development to provide state-of-the-art expression technologies
• Maintenance of frozen stocks of recombinant baculovirus
• Distribution of archived baculovirus stocks upon request
• Protein purification technology development
• Maintenance of genomic shRNA libraries/distribution of requested clones.