Study of bacterial gene expression heavily relies on E. coli-based in vivo and in vitro systems with RNA polymerase at their core. Although methods utilizing RNA polymerase assembled from individually expressed subunits or single-subunit expression vectors have met with considerable success, our experience indicates that co-overexpression vectors, featuring most or all RNA polymerase subunits have substantial advantages, particularly in efficiency of assembly of some mutants, deemed “defective” based on in vitro assembly attempts (see for example Artsimovitch et al, Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions, JBC, 2003, 278(14):12344-55). Here we present the latest co-overexpression vector for E. coli RNA polymerase, pVS10, and a sample protocol for purification of the enzyme produced using this vector (although it should be compatible with any classical RNA polymerase purification protocols as well).
pVS10 vector contains ORFs for 4 subunits, comprising E. coli core RNA polymerase rpoA (a), rpoB (b), rpoC (b’), and rpoZ (w). This plasmid has been successfully used as a platform for genetic manipulations of rpo genes and production of the wild-type as well as a number of mutant enzymes. It has been tested for expression in BL21(DE3), XJb(DE3), and DH5a(DE3) cell lines using both IPTG– and autoinduction (e.g. Overnight Express, Novagen) protocols. Yield varies with the toxicity of the construction, the identity of the strain and the induction conditions, usually within 1-10 mg/L of culture.
Induction and growth conditions:
The autoinduction using the Overnight Express (Novagen) reagents was carried out according to the manufacturer’s protocol with modifications to preculture preparation. BL21(DE3) cells transformed with pVS10 plasmid were inoculated from the frozen stock into 30 ml of LB medium (Miller) supplemented with ampicillin at 100 mg/L in 125 ml flask and grown with agitation at 37C till OD600 =0.6, when 0.6 ml of this culture were used to inoculate 600 ml of LB medium (Miller) supplemented with carbenicillin at 100 mg/L in 2 L flask, supplemented with Overnight Express reagents per Novagen protocol. The culture was incubated at 32C with agitation for 18 hours, and cells were harvested by centrifugation (4C, 5000 g, 10 min).
Lysis and Ni-affinity chromatography:
Cell pellet was frozen (-80C) and thawed (on ice), cells were resuspended in 50 ml of lysis buffer (500 mM NaCl, 50 mM Tris-HCl (pH 6.9), 5% glycerol, 0.2 mM b-ME) supplemented with 1X Complete EDTA-free protease inhibitors cocktail (Roche), and 1 mg/ml lysozyme. Suspension was incubated on ice for 60 min with occasional swirling, supplemented with Tween 20 to 0.2% and sonicated briefly to disrupt the cells. Extract was cleared from cell debris by centrifugation (27 000 g, 15 min, 4C). Cleared extract was combined with 4 ml of 50 % Ni-NTA slurry in lysis buffer and incubated with agitation for 30 min at 4C. The slurry was poured into a disposable gravity flow column (Bio-Rad) and drained. The column was washed with 20 volumes (40 ml) of lysis buffer, and 10 volumes of heparin column loading buffer (50 mM Tris-HCl, pH 6.9, 5 % glycerol, 0.5 mM EDTA, 1 mM b-ME) supplemented with 10 mM imidazole. Elution was carried out by adding 1 ml aliquotes of heparin column loading buffer, supplemented with 250 mM imidazole. Fractions with more than 0.2 mg/ml of protein (by Bradford) were analyzed using SDS-PAGE, the ones containing RNAP were pooled and if needed concentrated to total volume of 4-5 ml using Amicon Ultra 100K concentrators (Millipore).
RNAP-containing pool of fractions was loaded onto a 5 ml Heparin HiTrap column (Amersham) at 0.5 ml/min using low-pressure LC system (AKTAPrime, GE) (can use Heparin 16/10 columns for larger scale preps, load at 1 ml/min).
Column loaded with polymerase preparation was washed with 5 column volumes (25 ml) of loading buffer, elution was carried out using linear gradient of NaCl in loading buffer : 0-1.5 M NaCl over 120 ml at 1 ml/min. Peak fractions were collected, pooled, and dialyzed against loading buffer (alternatively the ionic strength can be reduced by several cycles of concentration-dilution with loading buffer using Amicon Ultra 100K concentrators).
Polymerase fractions collected in previous step were loaded onto 6ml ResourceQ column (GE) using the same system at 0.5 ml/min, washing and elution were carried out as described for Heparin Hi-Trap column. Peak fractions were analyzed by SDS-PAGE, pooled, and dialyzed against the DNA-agarose loading buffer (10 mM Tris-HCl (pH 7.9), 0.1 mM Na-EDTA, 0.2 mM b-ME, 5% glycerol).
Polymerase fractions collected in previous step were loaded onto 5 ml DNA-Agarose HiTrap column (GE) using AKTAPrime LC system at 0.2 ml/min. Loaded column was washed with 4 volumes (20 ml) of DNA-agarose loading buffer, and purified core RNAP was eluted using linear gradient of NaCl in loading buffer 0-1.5 M over 100 ml at 0.2 ml/min. Core RNAP containing fractions were analyzed by SDS-PAGE, pooled and dialyzed against RNAP storage buffer.
I received my Bachelors degree in microbiology from Southern Illinois University. As an undergraduate I worked in an independent food-testing laboratory assisting in research projects and performing routine testing. Working as a laboratory technician furthered my interest in microbiology and prompted me to attend graduate school. When searching for a university to attend, the balanced program at Ohio State stood out. The diversity of the professors, their backgrounds, and research made the program very attractive when compared to other schools. During a campus visit I met many of the professors and graduate students and was surprised by the overall friendliness and enthusiasm of the department. After I joined the department, lab rotations allowed me to explore various fields and interests as well as meet more of the students and professors.
In 2002 I joined the lab of Brian Ahmer. Currently I am working on the identification of genes regulated by SdiA, a LuxR homolog, in Salmonella typhimurium. One of the greatest advantages to being at a large university is access to equipment and facilities, that an individual lab could not support. The classes that I have taken are constantly under revision to include the most recent information and involve the students in the process. I am sure that the education that I receive from the department will serve me well in the future.
B.A. Biology, Gustavus Adolphus College, 1992
We are interested in how the ribosome works. The ribosome is a large (~2.5 MDa), two-subunit, RNA-based machine that translates the genetic code in all organisms.
In recent years, numerous X-ray crystal structures of the ribosome and its isolated subunits and many cryo-EM reconstitutions of functional ribosomal complexes have been reported. Today, a primary challenge for the field is to elucidate the functional roles of specific structural elements of the ribosome. Since the ribosome is the most common target of natural antibiotics, gaining a better understanding of how the ribosome functions should contribute substantially to the development of new antibiotics. In our group, we use mutagenesis and antibiotics to address questions of ribosome structure and function. One of our primary interests is translocation, the coupled movement of tRNA and mRNA within the ribosome. In recent work, we have shown that destabilization of the codon-anticodon helix accompanies movement of tRNA from the P/P to P/E state, an important transition during translocation.
|McGarry, K. G., Walker, S. E., Wang, H. and Fredrick, K. 2005. Destabilization of the P site codon-anticodon helix results from movement of tRNA into the P/E hybrid state within the ribosome. Mol. Cell 20: 613-622.
Abdi, N. M. and Fredrick, K. 2005. Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in E. coli . RNA 11: 1624-1632.
Yassin, A., Fredrick, K. and A. S. Mankin. 2005. Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics. PNAS 102: 16620-16625.
Noller, H. F., Hoang, L. and Fredrick, K. 2005. The 30S ribosomal P site: A function of 16S rRNA. FEBS Letters 579: 855-858.
Hoang, L., Fredrick, K. and H. F. Noller. 2004. Creating ribosomes with an all-RNA 30S subunit P site. PNAS 101: 12439-12443.
Fredrick, K. and H. F. Noller. 2003. Catalysis of ribosomal translocation by sparsomycin. Science 300: 1159-1162.
Fredrick, K. and H. F. Noller. 2002. Accurate translocation of mRNA by the ribosome requires a peptidyl group or its analog on the tRNA moving into the 30S P site. Mol. Cell 9: 1125-1131.
Noller, H. F., Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Lancaster, L., Dallas, A., Fredrick, K., Earnest, T. N. and J. H. D. Cate. 2001. Structure of the ribosome at 5.5 � resolution and its interaction with functional ligands . Cold Spring Harbor Symp. Quant. Biol. 66: 57-66.
Fredrick, K., Dunny, G. M. and H. F. Noller. 2000. Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30S subunits. J. Mol. Biol. 298: 379-394.
Immune mechanisms that determine outcome of “New world” cutaneous and visceral leishmaniasis.
The leishmaniases comprise several diseases caused by intracellular protozoan parasites belonging to Leishmaniaspecies leading to a wide spectrum of clinical manifestations and a global health problem. Among the parasitic infections, this disease is responsible for the highest number of DALYs (Disability adjusted life years; a measure of health burden) after malaria. “Old world” cutaneous leishmaniasis usually manifests as a localized self-healing skin lesion with long-term protective immunity in humans. In contrast, some forms of “New world” cutaneous leishmaniasis manifests as a chronic infection that is associated with mutilation of ear and disfiguring scars or as a severe mucocutaneous disease involving nasal and oropharyngeal mucosa with extensive tissue destruction. Visceral leishmaniasis is the most severe clinical form, characterized by hepatosplenomegaly, fever, abdominal pain and weight loss.
Our laboratory is interested in understanding the immune mechanisms that determine outcome of “New world” cutaneous and visceral leishmaniasis caused by L. mexicana and L. donovani respectively. We are particularly interested in studying the role of cytokines in regulation of immune responses during these two species of Leishmaniaand the use of cytokine and cytokine receptor gene deficient mice has been a very powerful tool in these studies. As cytokines can modulate functions of several cells of the immune system in vivo, we are now using cell-specific gene deficient mice lacking specific cytokine receptors on specific immune cells such as macrophages and T cells. These mice are generated using cre/lox technology that enables us to delete a gene in cell-specific manner. We believe that these studies will enable us to determine how cytokines regulate immune responses in vivo during leishmaniasis. With regards to L. donovani, our studies have focused on understanding the regulation of effector cell responses in murine visceral leishmaniasis caused by L. donovani. Of particular interest to our group is the determining the immune mechanisms that mediate protection and/or induce immunopathology during VL. More recently, in collaboration with the McGill University, we have initiated studies that focus on the development of amastigote-specific single candidate vaccine against visceral and “New world” cutaneous leishmaniasis that cause considerable morbidity and mortality in humans. Another area of research in our laboratory is understanding the immunological basis of gender-related differences in susceptibility to Leishmania. In these studies, we are interested in determining the roles of sex-hormones in modulation of immune response and determining the outcome of Leishmania infection. Our long-term goal is to identify the basic mechanisms by which cytokines regulate T cell responses and host immunity to cutaneous leishmaniasis caused by L. mexicana and visceral leishmaniasis caused by L. donovani and utilize this knowledge to develop a vaccine against these diseases.
Xu D, McSorley SJ, Tetley L, Chatfield S, Dougan G, Chan WL, Satoskar, AR, David JR, Liew FY. Protective effect on Leishmania major infection of MIF, TNF-alpha and IFN-gamma administered orally via attenuated Salmonella typhimurium. J. Immunol. 1998; 160: 1285-1289.
Satoskar AR, Khamis Al-Q, Alexander J. Sex-determined resistance against Leishmania mexicana is associated with the preferential induction of a Th1-like response and IFN-gamma production by female but not male DBA/2 mice. Immun Cell Biol. 1998; 76: 159-166.
Satoskar, AR, Okano M, Connaughton S, David JR, Labow M. Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice. Europ J Immunol.1998; 28: 2066-2074.
Stamm L, Raisanen-Solokowski A, Okano M, Russell M, David JR, Satoskar AR. Mice with STAT6-targeted disruption develop a Th1 response and control cutaneous leishmaniasis. J. Immunol. 1998; 161: 6180-6188.
Bozza M, Soares M, Bozza P, Satoskar AR, Brombacher F, Titus R, Shoemaker C, David JR. The selective PACAP-type I receptor agonist Maxadilan from Sand fly saliva protects mice against lethal endotoxemia. Europ J Immunol. 1998; 28: 3120-3127.
Bozza M, Satoskar AR, Lin G, Lu B, Humbles AA, Gerard C, David JR. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J. Exp. Med. 1999; 189: 341-346.
Satoskar AR, Stamm LM, Zhang, XM, Satoskar, AA, Okano, M, David JR, Terhorst C, Wang B. Mice lacking natural killer (NK) cells develop an efficient Th1 response and control cutaneous L. major infection. J. Immunol. 1999; 162:6747-6754.
Satoskar, AR, Stamm, LM, Zhang, X., Okano, M, David, JR, Terhorst, C., and Wang, B. NK cell deficient mice develop Th1-like response but fail to mount an efficient antigen-specific IgG2a antibody response. J. Immunol. 1999; 163: 5298-5302.
Stamm, LM, Satoskar, AA, Ghosh, S, David, JR and Satoskar, AR. STAT4-mediated IL-12 signaling pathway is critical for the development of protective immunity in cutaneous leishmaniasis. Europ. J. Immunol. 1999; 29:2524-2529.
Alexander, J, Satoskar AR and Russell, DG. Leishmania species: models of intracellular parasitism. J. Cell Sci.1999; 112:2993-3002. (Review).
Satoskar, AR, Rodig, S, Telford, SR, Satoskar, AA, Ghosh, S., von Lichtenberg, F., and David, JR. Interleukin 12 gene deficient mice are susceptible to L. donovani infection but have diminished hepatic immunopathology. Europ. J. Immunol. 2000; 30:834-839..
Monteforte, G., Takeda, K., Akira, S, David, JR and Satoskar, AR. Interleukin-18 is not critical for the development of Th1 response and control of cutaneous L. major infection. J. Immunol. 2000; 164: 5890-5893.
Alexander, J., Carter, KC., Al-Fasi, N., Satoskar, AR, and Brombacher, F. Effective drug therapy against visceral leishmaniasis is dependent on endogenous IL-4. Europ. J. Immunol. 2000; 30:2935-2943.
Hattori, H, Okano, M, Yoshino, T, Akagi, T, Nakayama, E, Saito, C, Satoskar, AR, Ogawa, T, Azuma, M and Nishizaki, K. Expression of co-stimulatory CD80/CD86-CD28/CD152 molecules in the nasal mucosa of patients with perennial allergic rhinitis. Clin. Exp. Allergy. 2001; 31:1242.
Okano, M, Azuma, M, Yoshino, T, Hattori, H, Nakada, M, Satoskar, AR, Harn Jr, DA, Nakayama, E, Akagi, T, and Nishizaki, K. Differential role of CD80 and CD86 molecules in the induction and the effector phases of allergic rhinitis in mice. Am. J. Res. Crtic. Care Med. 2001; 164: 1501.
Szabo, SJ, Sullivan, BM, Stemmann, C., Satoskar, AR, Sleckman, BP, and Glimcher, LH. Distinct effects of T-bet in Th1 lineage commitment and IFN-γ production in CD4 and CD8 T cells. Science. 2002; 295:338.
Greenwald, RJ, McAdam, AJ, Van der Woude, D, Satoskar, AR, and Sharpe, AH. Inducible co-stimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis. J. Immunol. 2002; 168: 991.
Rodriguez-Sosa, M, David, JR, Bojalil, R., Satoskar, AR, and Terrazas, LI. Susceptibility to the larval stage of the helminth parasite Taenia crassiceps is mediated by the Th2 response induced via STAT6 signaling. J. Immunol. (Cutting Edge) 2002; 168:3135.
Brown, JA, Greenwald, RJ, Scott, S, Schweitzer, A.N, Satoskar, AR, Chung, C, Schopf, LR, van der Woude, D, Sypek, JP, and Sharpe, AH. T helper differentiation in resistant and susceptible B7-deficient mice infected with Leishmania major. Eur. J. Immunol. 2002; 32:1764.
Hattori H, Okano M, Yamamoto T, Yoshino T, Yamashita Y, Watanabe T, Satoskar, AR, Harn DA, and Nishizaki K. Intranasal application of purified protein derivative suppresses the initiation but not the exacerbation of allergic rhinitis in mice. Clin. Exp. Allergy. 2002; 32:951.
Costa, CH, Stewart, JM, Gomes, RBB, Garcez, LM, Ramos, PK, Bozza, M, Satoskar, AR, Dissennayake, S, Santos, RS, Silva, MRB, Shaw, JJ, David, JR, and Maguire, JH. Asymptomatic human carriers of Leishmania chagasi. Am. J. Trop. Med. Hyg. 2002; 66:334-337.
Rodriguez-Sosa, M, Satoskar, AR, Calderon, R., Gomez-Garcia, L, Saaverda, R., Bojalil, R., and Terrazas, LI. Chronic helminth infection induces alternatively activated macrophages expressing high levels of CCR5 with low interleukin-12 production and Th2-biasing ability. Infect. Immun. 2002; 70:3656.
Parish, CL, Finkelstein, DI, Tripanichkul, W, Satoskar, AR, Drago, J, and Horn, MK. The role of Interleukin-1, Interleukin-6 and glia in inducing growth of neuronal terminal arbors in mice. J. Neurosci. 2002; 22:8034.
Wurster, AL, Rodgers, VL, Satoskar, AR., Whitters, MJ, Young, DA, Collins, M, and Grusby, MJ. Interleukin-21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naïve Th cells into interferon gamma-producing Th1 cells. J. Exp. Med. 2002; 196: 969.
Pien, GC, Nguyen, KB, Malmgaard, L, Satoskar, AR, and Biron, CA. A unique mechanism for innate cytokine promotion of T cell responses to viral infections. J. Immunol. 2002; 169:5827.
Rodriguez-Sosa, M, Rosas, LE, David, JR, Bojalil, R, Satoskar, AR*, and Terrazas, LI*. Macrophage migration inhibitory factor plays a critical role in mediating protection against the helminth parasite Taenia crassiceps. (*Joint senior co-authors) Infect. Immun. 2003; 71:1247.
Rodriguez-Sosa, M, Rosas, LE, Terrazas, LI, Lu, B, Gerard, C, and Satoskar, AR*. CC chemokine receptor 1 enhances susceptibility to Leishmania major during early phase of infection. Immunol. Cell Biol. 2003; 80:114.
Rodriguez-Sosa, M, Satoskar, AR, David, JR, and Terrazas, LI. Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps. Int. J. Parasitol. 2003; 33: 701.
Rosas, L, Keiser, T, Pyles, R, Durbin, J, and Satoskar, AR*. Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway. Eur. J. Immunol. 2003; 33: 1799.
Pan, J.H., Sukhova, G.K., Satoskar, A.R., David, J.R., Yang, J.T., Fu, H., Metz, C., Baugh, J.A., Bucala, R., Fang, K., Libby, P. and Shi, G.P. Regulation of cysteine protease expression by macrophage migration inhibitory factor. Circulation. 2004; 109:3149-3153.
Rodriguez-Sosa, M., Rosas, L.E., Saavedra, R., Satoskar, A.R. and Terrazas, L.I. STAT4-dependent IL-12 signaling pathway is required for resistance to the helminth parasite Taenia crassiceps. Infect. Immun. 2004; 71:1247-1254.
Wang, N., Satoskar, A.R., Faubion, W., Howie, D., Okamoto. S., Feske, S., Gullo, C., Clarke, K., Rodriguez Sosa, M., Sharpe, A.H. and Terhorst, C. SLAM controls T cell and macrophage functions. J. Exp. Med. 2004; 199:1255-1264.
Bhardwaj, N., Rosas, L.E., Lafuse, W.P., and Satoskar, A.R. Leishmania inhibits STAT1-mediated IFN-γ signaling in macrophages: Increased tyrosine phosphorylation of dominant negative STAT1β by Leishmania mexicana. Int. J. Parasitol. 2005; 35:7582.
Howie, D., Laroux, F.S., Morra, M., Satoskar, A.R., Rosas, L.E., Faubion, W.A., Julien, A., Rietdijk, S., Coyle, A.J., Fraser, C., and Terhorst, C. The SLAM family receptor Ly108 controls T cell and neutrophil functions. J. Immunol. 2005; 174:5931-5935.
Morra, M., Barrington, R.A., Abadia-Molina, A., Okamoto, S., Julien, A., Gullo, C., Kalsy, A., Edwards, M.J., Chen, G., Spolski, R., Leonard, W.J., Huber, B.T., Borrow, P., Biron, C.A., Satoskar, A.R., Carroll, M.C., and Terhorst, C. Defective B cell responses in the absence of SH2D1A. Proc. Natl Acad. Sci. USA. 2005; 102:4819-4823.
Okano, M., Hattori, H., Yoshino, T., Sugata, Y., Yamamoto, M., Fujiwara, T., Satoskar, A.A., Satoskar, A.R., and Nishizaki, K.Nasal exposure to Staphylococcal enterotoxin enhances the development of allergic rhinitis in mice. Clin. Exp. Allergy. 2005; 35:506-514.
Powell, N.D., Papenfuss, T.L., McClain, M.A., Gienapp, I.E., Shawler, T.M., Satoskar, A.R., and Whitacre, C.C. Macrophage migration inhibitory factor is necessary for progression of experimental autoimmune encephalomyelitis. J. Immunol. 2005; 175:5611-5614.
Rosas, L.E., Barbi, J., Lu, B., Fujiwara, N., Gerard, C., Sanders, V.M., and Satoskar A.R. CXCR3-/- mice mount an efficient Th1 response but fail to control L. major infection. Eur. J. Immunol. 2005; 35:515-523.
Rosas, L.E., Keiser, T., Barbi, J., Satoskar, A.A., Septer, A., Kcazmarek, J., Lezama-Davila, C.M., and Satoskar, A.R. Genetic background influences immune responses and disease outcome of cutaneous L. mexicana infection in mice. Int. Immunol. 2005; 17:1347-1357.
Hattori, H., Okano, M., Kariya, S., Nishizaki, K. and Satoskar, A. R. CD40-CD40L interaction is involved in pathogenesis of SEA induced allergic rhinitis. Amer. J. Rhinol. 2006; 20:165-169.
Liang, S.C., Greenwald, R.J., Latchman, Y.E., Rosas, L., Satoskar, A. R, Freeman, G.J. and Sharpe, A.H. PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis. Eur. J. Immunol. 2006; 36:58-64.
Reyes, J., Terrazas, L.I., Espinoza, B., Gomez-Garcia, L., Cruz-Robles, D., Rivera-Montoya, I., Snider, H., Satoskar, A. R. and Rodriguez-Sosa, M. Macrophage migration inhibitory factor (MIF) plays a critical role in host defense against acute Trypanosoma cruzi infection. Infect. Immun. 2006; 74:3170-3179.
Rosas, L.E., Barbi, J., Snider, H., Satoskar, A.A., Lugo-Villarino, G., Keiser,T., Papenfuss, T, Durbin, J, Radzioch, D, Glimcher, LH and Satoskar, A. R. Cutting edge: STAT1 and T-bet play distinct roles in determining outcome of visceral leishmaniasis caused by Leishmania donovani J. Immunol. 2006; 177:22-25.
Rosas, L.E., Satoskar, A.A., Roth, K., Keiser, T., Barbi, J., Hunter, C.A, de Sauvage, F. and Satoskar, A. R. IL-27R (WSX-1/TCCR) gene deficient mice display enhanced resistance to Leishmania donovani infection but develop severe liver immunopathology. Am. J. Pathol. 2006; 168:158-169.