Protein Expression Facility

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

The Protein Expression Facility (PEF) is part of the Genome Integrity and Structural Biology Laboratory. The mission of the PEL is to helps NIEHS researchers design projects to express, purify, and biophysically characterize proteins and macromolecular complexes. In 2025, the PEF mission was expanded to include trans NIH projects through the collaborations with the Cryo-Em core. The PEF can perform or assists in all aspects of protein expression and purification projects, from construct design and protein purification through structure function studies. The structural work is performed in close cooperation with both the Cryo-EM and X-ray crystallography cores both located in the GISBL. The PEF group members work directly with investigators to determine how best to meet their protein needs and to assure a seamless transfer of materials and methods as well as providing scientific and technical assistance. The approach that the PEF uses with each new protein expression project reflects the understanding that there is no apriori means of determining which expression system will work for any given new protein. Gateway cloning technology is used to rapidly generate expression vectors to express native as well as N and/or C terminal tagged proteins in E. coli, baculovirus/insect cells, and mammalian cells. Although Gateway vectors are our first option with a new protein target, we will work with any expression vector/system that the investigator groups want to use. The effects of different expression cell lines, growth media, expression temperatures, and co-expression with molecular chaperones on protein folding and activity are routinely tested. Protein refolding techniques using rapid dilution and high hydrostatic pressure (Barofold) are available for proteins that only or mostly express incorrectly folded. In addition to the above techniques, the protein expression group is continually working to add vectors for Lenti viral generation, and BacMam (baculovirus mediated expression in mammalian cells), into our Gateway vector collection. Lenti viruses are generated by the Viral Vector Core (VVC). The protein expression facility (PEF) functions as a support group for the principal investigators at the NIEHS. In 2025, the PEF was charged with supporting trans-NIH projects through collaborations with the Cry0-EM core. 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. Project examples: Allergenic protein initiative: 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: 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. During the last year, more than 20 anti-bodies were expressed to support the allergenic studies. The yield of antibody ranges from 100 to 500 mg/L of CHO cells, these antibodies 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 with the Wade Group 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 has demonstrated the expression of multiple fluorescent proteins simultaneously. This vector system is currently being used for several trans-NIH projects involving the Cryo-EM Core as well. Expression of Alpha7 nicotinic acetylcholine receptor and the Yakel group. The PEF expressed the integral membrane alpha 7 nicotinic acetylcholine receptor using both transient expression using bacmam vectors and a stable HEK293-f cell line. This required the co-expression of the chaperone protein called Nacho. Once expression was established, the PEF then worked out a purification for the alpha-7 receptor using the protein SAP-A (produced by the PEF). The purified Alpha 7 protein’s structure was determined by Cry0-EM with several protein complexes. Expression of protein standards for Cryo-EM studies and the Borgnia Group: The PEF has produced both the 20S proteasome 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. Covid-19 projects The PEF currently has projects limited to generation of various soluble Spike proteins for various projects that NIEHS investigators are currently working on. Trans NIH projects in collaboration with the Cryo-Em Core, Borgnia Group. There have been 3 main trans NIH projects (Ghrelin GPCR complex, Zinc transporter 3, and TLR1/2-CD206 complex). All 3 projects were successfully able to demonstrate expression in the desired system. The PEF is currently working on purification of the transmembrane proteins for structure function studies.