The protein expression group 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 large quantities 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 five different n-terminal tags (6 x His, 6 x His-Thioredoxin, 6 x His-glutathione S-transferase, 6 x His-Maltose binding protein, 6 x His-NusA). Initial expression test are done in Rosetta2(DE3)pLacI cells at 18 C and 30 C. 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. This is not the only cell line that does this, and we are not endorsing this cell line over the others. 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 is tried first. Expression is investigated using three n-terminal tags (6 x His, 6 x His-glutathione S-transferase, and 6 x His-Maltose binding protein) and two cell lines (SF9 and High Five). Expression trials are carried out at both 27 C and 20 C. All new baculovirues that are generated are titered using the Sf9et cells to demonstrate infectivity as well as determining viral titer. Once the system is established and future scale up is done using TIPs protocol to save time and storage conditions. The protein expression group also has vectors available for expression of protein in mammalian cell lines. Expression is tested in Cos-7, HEK293, CHO and 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. If the above methods fail to yield a protein that in other that insoluble aggregates (inclusion bodies), then protein refolding is 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 Project: 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 on the same mRNA. 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. Allergenic protein initiative: Robert London 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 (Der p 5, 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, lipopolysachrides 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. hPol Nu project: Thomas Kunkel Human DNA polymerase Nu (Pol Nu) is a conserved family A DNA polymerase of unknown biological function. Physical and biochemical characterization aimed at understanding Pol Nu function is somewhat hindered by the fact that when over expressed in E. coli, Pol Nu is largely insoluble, and the small amount of protein that is soluble is difficult to purify. To obtain soluble Pol Nu for future studies, high hydrostatic pressure was used to solubilized and refold active Pol Nu from inclusion bodies. Active Pol Nu can be refolded and purified. The properties of the refolded enzyme are comparable to those of the small amount of Pol Nu that can be purified from the soluble fraction. The approach used here may be applicable to other DNA polymerases that are insoluble when expressed in E. coli.
|Perdivara, Irina; Petrovich, Robert; Allinquant, Bernadette et al. (2009) Elucidation of O-glycosylation structures of the beta-amyloid precursor protein by liquid chromatography-mass spectrometry using electron transfer dissociation and collision induced dissociation. J Proteome Res 8:631-42|