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 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. In all cases, co-expression of green fluorescent protein driven by a different promoter is used to help titer the baculovirus and demonstrate that infection was successful. 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: Acetylcholine-binding Protein Project: Jerrel L. Yakel and Robert E. London We identified a homolog of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella capitata, from the annelid phylum. The Capitella capitata AChBP (cc-AChBP) has 21-30% amino acid identity with known molluscan AChBPs. Sequence alignments indicate that cc-AChBP has a shortened cys-loop compared to other cys-loop receptors, and a variation on a conserved C-loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D-loop of cc-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand-binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 of cc-AChBP is glycosylated when expressed using HEK293 cells. Small angle X-ray scattering (SAXS) data are consistent with cc-AChBP existing as a soluble pentamer in solution. SAXS models for the apo-protein and nicotine-bound forms of the pentamer indicate large conformational changes of the protein upon binding of the ligand;these alterations appear not to be limited to just the C-loop/F-loop region. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to cc-AChBP with high affinity, with KD values of 28 uM, 200 nM, and 100 nM, respectively. Choline bound with a lower affinity (KD = 165 uM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may possess variation in hallmark motifs such as ligand-binding residues and cys-loop length, and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca. The core facility prepared a stable cell line to produce the cc-AChBP. The production was then scaled up so that mgs of protein could be produced. A purification protocol was also developed by the core facility. This is an ongoing project that also involves the Protein Microcharcterization Core Facility and the collaborative crystallization group. Rhox13 project: Edward M Eddy Homeobox genes encode transcription factors whose expression organizes programs of development. A number of homeobox genes expressed in reproductive tissues have been identified recently, including a colinear cluster on the X chromosome in mice. This has led to an increased interest in understanding the role(s) of homeobox genes in regulating development of reproductive tissues including the testis, ovary, and placenta. The Eddy group identified and characterized a novel homeobox gene of the paired-like class on the X chromosome distal to the reproductive homeobox (Rhox) cluster in mice. Transcripts are found in the testis and ovary as early as 13.5 days post coitum (dpc). Transcription ceases in the ovary by 3 days post partum (dpp), but continues in the testis through adulthood. The Rhox13 gene encodes a 25.3 kDa protein expressed in the adult testis in germ cells at the basal aspect of the seminiferous epithelium. The protein expression group expressed the Rhox13 gene in E.coli and purified it. This protein was used to confirm the identity of the protein in the native cells used in these studies. 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.
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