Protein Expression Facility

National Institute Of Environmental Health Sciences

Investigators

Linked publications & trials

Abstract

The protein expression facility (PEF) functions as a support group for the principal investigators at the NIEHS. Our focus is to provide the research groups with a means to generate the proteins they require 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, and expressing monoclonal antibodies. 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 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 for 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 27C and 20C. 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 principal investigator. Expression is first tested transiently followed by the generation of a stable cell line, if this is possible/desired. 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: 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 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. Antibody interaction with Ara h2: Robert London, Geoff Mueller, 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 sent to the NIEHS investigators for structure function studies (how do the antibodies differ in their interaction with Ara h2). The expiCHO-s system is now used routinely to express multiple antibodies for this project. The yield of antibody ranges from 100 to 500 mg/L of CHO cells, this year alone multiple grams of antibody have been produced to support the NMR, x-ray crystallography and Cryo-EM studies involved in these allergenic protein project. 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. The PEF previously demonstrated the ability to express the 5 protein NuRD complex in support of the Wade group. Building on this, the PEF has undertaken the construction of vectors similar to those for the Macrobac in insect cells for use with mammalian cells. These MacroBacMam vectors are nearing completion. At this time, we have demonstrated that the vectors will express proteins in mammalian cells and can be used to make bacmids using DH10Bac cells. The PEF hopes to demonstrate the expression of multiple fluorescent proteins simultaneously this fall and begin construction of MacroBacMam viruses for the Nurd complex and several others complexes soon after that. Expression of protein standards for Cryo-EM studies and the Borgnia Group: The PEF has produces both the 20S proteasome from Thermophilus acidophilum and apo-Ferritin as test molecules for the new Cryo-EM facility at the NIEHS. Both proteins are currently being used to help people learn how to prepare grids, collect data, and solve structures using Cryo-EM. The apo-Ferritin structure has been refined to 3.1 angstrom resolution on the Arctica instrument. The PEF is working on using the apo-Ferritin as scaffold molecule for to display small proteins for Cryo-EM structure determination. Covid-19 projects The PEF currently has all the orfs for the original Covid19 virus in expression vectors on hand. We provide them to any investigator at the NIEHS who wants them. Our primary focus has been to support the Viral Vector Cores efforts to make pseudo-virus particles for test virus infection models. The NIEHS does not have the BL3 facilities to study the actual Covid-19 virus. The PEF also has been expressing and purifying the soluble version of the spike protein and the RBD of the spike protein for various structure function initiatives (NMR, x-ray crystallography, Cryo-EM). In a collaborative effort with several groups outside the NIEHS, the PEF has expressed and purified nanobodies raised against the RBD of the spike protein. The PEF currently has the genes from the spike protein from the original Covid 19, the D614G mutant, the Alpha variant (B1.1.7, UK variant), Beta variant (B1.351, South Africa variant), Delta variant (B1.617.2, India variant), Gamma variant (P1, Brazil variant). There are several Cryo-EM structures of the different variant spike proteins with several showing interaction with different nanobodies. At least one of the nanobodies had its x-ray crystal structure solved. Crystallographic studies of several versions of the RBD with and without nanobodies are underway as well.