The protein expression facility (PEF) functions as a support group for the principle investigators at the NIEHS. Our focus is to provide the investigative groups at the NIEHS with a means to generate the protein of interest so that they can perform their experiments. These projects range from generating enough of a given protein to perform structure function studies, to the generation of protein fragments for anti-body generation, to creating stable cell lines for more in vivo assays. Each new protein that is expressed in E. coli is tested using four different n-terminal tags (6 x His, 6 x His-Thioredoxin, 6 x His-glutathione S-transferase, and 6 x His-Maltose binding protein). Initial expression testing is done in Rosetta2(DE3) pLacI cells at 18C and 30C. These tags help to fold/solubilize the protein of interest and provide a uniform initial purification step that helps to identify which combination of tag and temperature yield the most of the desired product. The Rosetta cell line helps remove any codon bias against expression in E. coli. Once expression is demonstrated and a tag selected other variables such as alternative cells lines, media and temperature can all be optimized to give the best yield. If expression in E. coli fails to work or is not feasible due to the need for post translational modifications, then baculovirus/insect cell expression system is tried. Expression is investigated using three n-terminal tags (6 x His, 6 x His-glutathione S-transferase, and 6 x His-Maltose binding protein) using two cell lines (SF9 and High Five). Expression trials are carried out at both 27 C and 20 C. All new baculovirus that are generated are tittered using the Sf9et cells to demonstrate infectivity as well as determining viral titer. Once the system is established, all future scale up is done using TIPs protocol to save time, storage space, and provide for long term storage of the baculovirus at -135C. The PEF also has vectors available for expression of protein in mammalian cell lines. Expression is tested in Cos-7, HEK293, CHO and/or Hela cells unless a more unique cell line is desired by the principle investigator. Expression is first tested transiently followed by the generation of a stable cell line, if this is possible/desirable. If the above methods yield a protein that is only in insoluble aggregates (inclusion bodies), then protein refolding can be attempted. Each refolding project is tested using both rapid dilution and a high hydrostatic pressure approach. The rapid dilution approach is performed in a 96 well multi matrix format while the high hydrostatic approach uses a more sequential multi-sample (20 sample per run) approach. Project examples: Glis 1,2,3 and JazF1 Projects: Anton Jetten Expression of full length Glis 1, 2, or 3 in mammalian cells proved to be lethal events. This unforeseen eventuality precluded the use of pull down experiments to identify binding partners. Even the expression of domain fragment proved difficult as all cell lines tested would shut down the expression of the Glis fragment over time. Large scale transient expression proved ineffective and costly. To get around these factors, fragments of Glis 1, 2, or 3 as well as JazF1 were expressed using IRES vectors that co-expressed eGFP. Stable cell lines were made using a selectable marker in conjunction with cell sorting for expression of eGFP. This way cells that stopped expressing the protein of interest but maintained viability in the present of the selectable marker could be eliminated. This approach proved successful in generating sufficient number of cells expressing the protein of interest to do the pull down experiments to identify binding partners. A second approach using the Tet on system was also used. Fragments of Glis 1, 2, and 3 were expressed using a tetracycline inducible promoter. Stable HEK293-f cell were generated and up to 2L of each stable cell were grown and handed off to the Jetten group for binding partner identification. Allergenic protein initiative: Robert London and Geoff Mueller The NMR group has begun a series of structure function studies on a group of proteins which are known allergens. To support this effort, the PECF has had the genes of target proteins (Ara h2, Bla g2, Bla g6, Der p 2, Der p5, Der p 7, Scfv, Can f 5, Cat R 1, Rage) synthesized (this saves time, money, and effort on our part) for expression in E. coli and baculovirus. Expression in E. coli proved sufficient in all cases to obtain enough protein for x-ray crystallography and NMR structure studies; however, lipopolysaccharides derived from the bacteria complicated functional analysis in the allergen assays. In order to pursue this line of study, the proteins were express and purified from insect cells using baculoviruses. Fms Related Tyrosine Kinase 3 ligand (FTL3-l): Don Cook and Jennifer Martinez FTL3-l was requested by two different groups in the IIDL (Immunity, Inflammation, and Disease Laboratory). This protein is generally used in cell based assay to look at innate immunity response in dendritic cells. Both groups needed the protein to use as a reagent in their studies. The PECF had the gene synthesized for expression in E. coli. We then worked out a refolding protocol that yielded mgs of active protein that was endotoxin free for use in their cell assays. This amount of protein allows both groups to proceed with their studies over lengthy periods of time without batch to batch variations as they had seen in commercial sources. Antibody interaction with Ara h2: Robert London, and Lars Pederson Ara h2 is one of the proteins involved in peanut allergies. Investigators at the NIEHS are collaborating with allergy physicians to look at why some patients respond to new immune therapies to Ara h2 allergies and some do not. To examine this several antibodies to Ara h2 were isolated from patients and then 5 were selected based on response groupings to the therapy. These antibodies were send to the NIEHS investigators for structure function studies (how do the antibodies differ in their interaction with Ara h2). The PECF already had the Ara h2 protein expressed and purified (see above). The 5 different antibodies expression was demonstrated using HEK293-f cells. Expression was scaled up using suspension cultures and transient transfection using PEI max. This system allows for the production of antibody in excess of 50 mgs so far, enabling crystal trials of the antibody-Arah2 complex. Mammalian expression of proteins for the Stanley Group The Stanley Lab frequently utilizes the support of the Protein Expression Core/Structural Biology Core for assistance with mammalian cell culture including large scale protein expression and small scale co-immunoprecipitations. For example, to characterize the association between the essential AAA-ATPase NVL2 and the ribosome assembly factor WDR74 the Core performed a series of transient transfections of different truncations and variants of these two proteins. Co-immunoprecipitations with the cores in-house made GFP resin were then used to assay for protein association. Using this methodology, the Stanley lab was able to determine a unique method of association of the AAA-ATPase motor protein and its substrate. Expression of large protein complexes for the Wade Group The PEF is determining the optimal method for expression of the NuRD complex. Each of the target proteins have been expressed using single target baculoviruses. Mixing the single target baculovirus failed to yield enough of the complex as desired by the Wade group. The PEF is currently evaluating the use of the MultiBac, MacroBac, and Gibson Assembly methods to generate baculoviruses the express up to 8 different components at once. The PEF has worked on 68 different projects for 21 diff
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