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Protein Expression and Purification Core Facility (PEP)
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PEP Director:
Michael Miley, Ph.D.
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Medical Research Building B
CB# 7365
tel: +1-919-843-7174

Services

The facility offers a full range of protein expression and purification (PEP) services. Outlined below is a general description of the services and capabilities that we offer. We are co-localized with the macromolecular x-ray crystallography (MX) core, so modifying your project to include crystallography is really easy. We, however, do not offer any standardized, fixed price services like the MX core; all PEP projects present their own unique set of challenges and goals. Each project will be billed as close as possible to actual costs (non-profit, as we are a recharge center); this will include all consumables and staff time required.

We also allow and encourage users with prior experience to use the facilities autonomously. To do so, users must undergo training and/or be certified as an independent operator by our staff. Training time typically varies with each user and activity.

Our primary mission is to provide services to UNC users, but we also offer our services to external users including non-UNC academic users and commercial companies.


Expression Services

The facility currently utilizes E. coli , insect cell (baculovirus mediated), and mammalian cell expression systems. We have a library of expression vectors that users have access to for their projects. Typically, users provide a sequence-verified expression vector containing the gene(s) of interest. Alternatively, our staff can undertake expression vector construction if requested.

E. coli Expression

  • Small-scale test expressions in deep-well blocks can be conducted to determine optimal expression conditions

  • Large multi-liter shaking, suspension cultures of E. coli can be grown and processed at temperatures ranging from 18°C to 42°C. Max capacity is 18L at a time

  • 10L fermentation runs using our New Brunswick SIP Bioflow 415 instrument can be conducted when large quantities of cell mass are needed

  • The facility stocks a variety of E. coli strains to address specific issues like rare-codon usage, protein mis-folding, and expression in specialized media, for example, for the production of seleno-methione variants or 13C and 15N labeling


Baculovirus-Mediated Insect Cell Expression

  • Generation, titer determination, and storage of recombinant baculovirus

  • We grow and maintain both Sf9 and Hi-five insect cell lines

  • Small-scale test expressions in both suspension and adherent cultures can be conducted to determine optimal expression conditions

  • The facility has the capacity to carry out multi-liter, shaking suspension culture expression runs, max ~20L


Mammalian Cell Expression

  • We grow and maintain three main suspension cell lines cultured in minimal media: HEK 293-F (freestyle), CHO-S (freestyle), NS0.

  • We have a number of adherent cell lines that are used for specialized circumstances like limiting glycosylation or enabling DHFR stable cell selection.

  • We are able to generate stable, clonal cell lines expressing your protein of interest. We also have several strategies that we can use to optimize expression levels in these systems. 

  • Small-scale test expressions in both suspension and adherent cultures via transient transfection can be conducted to determine optimal expression conditions.

  • We have the capacity to carry out large scale expression experiments (suspension and adherent), Max ~15L of suspension culture and ~3L of adherent culture in roller bottles.


Protein Purification

The facility has all the relevant materials, instrumentation, and expertise to accommodate all popular purification methods and systems; from basic gravity columns and dialysis to high end FPLCs and Tangential flow filtration. If users need to utilize a technique and/or system that is not present in the facility we will work with you to try and get it working here.


Fast Protein Liquid Chromatography (FPLC)

The facility is equipped with FPLCs and a wide assortment of both high- and low-grade chromatography columns, covering the vast majority of protein-purification needs (affinity capture/ion exchange/gel filtration). In addition, users may supply their own columns for specialized projects.

AKTAxpress – We have 2 of these instruments. They are advanced chromatography systems designed for automated, multistep protein purification of both single and multiple samples.

AKTA FPLC – We have one of these. It is an older workhorse system that is ideal for developing a purification protocol at small scale before transitioning to the AKTAxpress systems.

  • Fee – FPLC time rate.

Tangential Flow Filtration (TFF)

TFF is a process for the rapid concentration and dia-filtration (buffer-exchange) of large volumes of solutions, typically in preparation for further purification steps. Our system can accommodate volumes up to 10L and proteins with molecular weights of 10KDa and larger.

  • Fee – Tangential flow filtration rate


General PEP Laboratory Use

The facility has a fully functional laboratory equipped to facilitate routine protein expression, purification, and molecular biology experiments as well as their documentation. The laboratory is available for general use by individuals to complement the capabilities of their own laboratories. In addition to the activities listed above, a variety of standard activities can be carried out, such as E. coli transformations, gel electrophoresis/western blotting, small-volume protein concentration, PCR, cloning and other general molecular-biology tasks, limited proteolysis experiments, etc.

  • Fee – Consumables and service rates if applicable


Training, Expertise, and Consultation

Highly trained scientists in the CSB are available to

  • provide expert training to users, affording them the opportunity to acquire valuable general know-how in protein-expression and purification

  • help design and implement PEP strategies in a project-specific manner

  • provide assistance with data analysis and interpretation

  • to independently carry out PEP projects on the user’s behalf

  • participate in grant writing and preparations of figures for publication and presentation

  • Fee – Staff time rates


Conferences

 

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