The Protein Expression and Libraries Facility (Protein Expression) provides Cancer Center members efficient and expert technical assistance in recombinant DMA plasmid engineering, protein expression in bacteria and baculovirus-infected insect cells, purification of recombinant proteins to homogeneity, and production of high-titer stocks of retroviruses (e.g. lentiviruses) for delivery of shRNA and cDNAs to mammalian cells. The Facility has expertise in all aspects of vector technology for protein expression, baculovirus generation, recombinant protein expression, and affinity and conventional chromatography approaches to protein purification, and production of infectious lenti-/retroviruses. The proteins produced by the facility must be of high quality and purity in order to be used by Cancer Center scientists to achieve a wide range of experimental objectives, such as characterization of enzymatic activities, crystallization for structural analysis, characterization of structure-function relationships of protein:protein, protein:nucleic acid, and protein:small molecule interactions;development of assays for small molecule high throughput screening;and immunization of mice to generate custom antibodies utilizing the Hybridoma Facility. The facility will continue to expand it's repertoire of vectors available for protein expression, included high titer retroviral vectors, acquire RNAi libraries, and implement a new assay development service for highthroughput screening of small molecule libraries. The facility personnel are highly trained technical experts in all areas of the proposed support services to be provided to the individual projects. The centralization and standardization of these practices under the direction of very experienced staff allows for high-throughput expression plasmid construction and large-volume protein expression services, including quality assurance and control procedures to ensure efficient, consistent production and purification of high quality recombinant proteins. The Facility also maximizes biosafety by confining retrovirus (e.g. lentivirus) production to a centralized biosafety level 2 (BSL2) unit, which prevents aerosolization of viruses from contaminating incubators and parental cultures of cell lines, and improves quality control in the production of virus stocks to be used in gain- and loss-of-function experiments in vitro and in vivo. Currently the Facility is being expanded to accommodate the storage and handling of viral libraries, additional purification instruments, and the rapid assay systems to meet Cancer Center needs.

Public Health Relevance

Recombinant DMA technology has provided the unique opportunity to produce otherwise rare proteins derived from recombinant genes. The availability of these proteins has enabled many types of experiments that would have otherwise been impossible. Also, the facility will provide and manage libraries of shRNA genes in lenteviral vectors that can be used to manipulate the expression of genes for cancer research.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Center Core Grants (P30)
Project #
5P30CA010815-44
Application #
8461268
Study Section
Subcommittee G - Education (NCI)
Project Start
Project End
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
44
Fiscal Year
2013
Total Cost
$148,129
Indirect Cost
$62,000
Name
Wistar Institute
Department
Type
DUNS #
075524595
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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