The Protein Expression Facility is a shared resource laboratory that provides Wistar Cancer Center Members and non-Wistar scientists technical assistance with viral vector preparation and the expression and purification of recombinant proteins. The Facility has greater than 20 years of experience in recombinant protein expression with special expertise in the use of baculovirus expression systems (BVES). The Facility offers the following services:
1. Recombinant plasmid DNA engineering
2. Viral vector production (i.e. baculovirus and retrovirus)
3. Analytical and preparative scale expression of nascent or epitope-tagged recombinant proteins
4. Protein purification
These goals are accomplished by a centralized laboratory with dedicated, experienced staff, which enables high-throughput, economy of scale, virus preparation and protein expression services, including quality assurance and control procedures to ensure efficient, consistent production and purification of recombinant proteins and viral vectors. Many recombinant proteins produced by the facility have been used for crystallization efforts, analytical biochemistry studies designed to investigate enzymatic properties, structure-function relationships between protein-protein, protein-nucleic-acid, and protein-small molecule interactions, custom antibody production, experimental cancer vaccines, and development of miniaturized assays for small molecule screening.
The facility is supported in part by an NCI Cancer Center Support Grant and a grant from the NIH National Institute of Aging (PO1 AG031862).
"The facility staff will assist users in designing a cloning strategy to engineer an appropriate recombinant plasmid DNA that meets the specific goals of the project. The facility supports conventional, ligation independent, Gateway, and site-directed mutagenesis cloning technologies. The nucleic acids (e.g., cDNA, promoter, shRNA, etc.) are isolated from a customer-supplied plasmid DNA(s) and subcloned into a new vector of choice. Newly derived plasmid DNAs are authenticated by restriction endonuclease digestion, DNA sequencing, and prepared for subsequent transfection based applications."
"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 in bacteria.
The core maintains a repository of inducible bacterial expression vector technologies. This collection of expression vectors allows 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 maltose binding protein (MBP) 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 (e.g. Rosetta (DE3))."
Services include: optimization of soluble protein expression and preparative production or recombinant proteins.
"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 has >20 years of experience in preparing baculovirus vectors from nearly all commercially available BVES.
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 include: preparation of high-titer baculovirus stock, amplification of high-titer baculovirus stocks, analytical scale productions for optimization of protein expression, and preparative scale productions."
"The Facility provides analytical and preparative scale, one- or two-step purification of recombinant proteins expressed in bacteria and baculovirus infected insect cells. 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, amylose-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."
"The development of retroviral delivery systems for mammalian cells has greatly enhanced the ability of scientists to deliver genetic material to cells of diverse origin in order to study the role of a particular protein or network of proteins in a specific cell type or biological process. The goal of the retrovirus production unit is to assist Cancer Center members with the preparation 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 preparation process. The Facility provides a variety of services including prepackaged vector preparations, custom vector packaging, and vector titrations.
The Protein Expression Facility’s work with recombinant retrovirus DNA is registered with the Wistar Institute IBC (approval #: 21102382, exp. 3/2014)"