A novel cell-free protein synthesis system

Sitaraman, Kalavathy; Esposito, Dominic; Klarmann, George J.; Grice, S F Le; Hartley, James L.; Chatterjee, Deb

doi:10.1016/j.jbiotec.2004.02.014

Cited by 120 publications

(90 citation statements)

References 19 publications

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“…1,250 bp product that was used as a template for OCFS. Because linear DNA templates are susceptible to degradation by the ExoV nuclease activity of RecBCD, we added an inhibitor of this activity, the λ bacteriophage GamS protein, 23 for efficient high-yield protein expression (Figs. S1 and S2).…”

Section: Resultsmentioning

confidence: 99%

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Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

Yin¹,

Garces²,

Yang³

et al. 2012

mAbs

129

157

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Section: Resultsmentioning

confidence: 99%

“…28 The resulting single band PCR fragments were purified using PureLink TM 96 PCR Purification Kit (Invitrogen) and quantitated using PicoGreen (Invitrogen, Carlsbad, CA). Based on the titration of linear DNA, an average of >30 nM linear template DNA with 1 μM GamS, 23,45 was used for each cell-free reaction in the 96-well plate.…”

Section: Discussionmentioning

confidence: 99%

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

Yin¹,

Garces²,

Yang³

et al. 2012

mAbs

129

157

View full text Add to dashboard Cite

show abstract

“…Lysates were produced from E. coli BL21(DE3) cells induced with IPTG (ref. 37 and references therein). Expression reactions contained the following components: 40% (vol/vol) lysate; 230 mM potassium glutamate; 58 mM Hepes (pH 7.5); 2% (wt/vol) PEG 8000; 80 mM ammonium acetate; 12 mM magnesium acetate; 1.2 mM ATP; 0.8 mM UTP, CTP, and GTP; 0.65 mM cAMP; 1.7 mM DTT; 34 g/ml folinic acid; 30 mM 3-phosphoglycerate; 0.17 mg/ml E. coli tRNA; 4.5 mM SeMet; 2 mM each of the other 19 aa; and 50 g/ml DNA template.…”

Section: Methodsmentioning

confidence: 99%

X-ray structure of EmrE supports dual topology model

Chen

Pornillos

Lieu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

263

277

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EmrE, a multidrug transporter from Escherichia coli, functions as a homodimer of a small four-transmembrane protein. The membrane insertion topology of the two monomers is controversial. Although the EmrE protein was reported to have a unique orientation in the membrane, models based on electron microscopy and now defunct x-ray structures, as well as recent biochemical studies, posit an antiparallel dimer. We have now reanalyzed our x-ray data on EmrE. The corrected structures in complex with a transport substrate are highly similar to the electron microscopy structure. The first three transmembrane helices from each monomer surround the substrate binding chamber, whereas the fourth helices participate only in dimer formation. Selenomethionine markers clearly indicate an antiparallel orientation for the monomers, supporting a ''dual topology'' model. membrane protein structure ͉ multidrug transport ͉ SMR family O ne of the mechanisms by which cells neutralize the effect of toxic compounds is through the action of membrane transporters. Secondary transporters, such as the EmrE protein of Escherichia coli, couple the efflux of drugs to the inward movement of protons across the cell membrane (refs. 1 and 2, and references therein). EmrE is the prototypical member of the SMR (small multidrug resistance) family and is one of the smallest known transporters in nature, composed of only 110 amino acid residues. Studies have shown that the basic functional unit of EmrE is an oligomer, as would be expected for a membrane protein of its small size. It appears established that the basic functional unit of EmrE is a homodimer, as shown by oligomerization assays, substrate binding experiments, negative dominance studies, and crosslinking analyses (3-8). This conclusion is further supported by the existence of paired SMR proteins, such as YdgE/YdgF of E. coli, and EbrA/EbrB and YkkC/YkkD of Bacillus subtilis. These transporters require coexpression of the two component polypeptides for proper drug efflux activity and presumably form heterodimers analogous to the EmrE homodimer (9-13).Electron microscopy (EM) studies of EmrE in complex with a transport substrate, tetraphenylphosphonium (TPP), and reconstituted in lipid bilayers have revealed the overall architecture of the transporter at 7.5 Å resolution in-plane and 16 Å perpendicular to the membrane (14). EmrE binds TPP as an asymmetric dimer, in a chamber that appears open to one side of the bilayer. Each monomer is composed of four transmembrane (TM) helices. Because of the low resolution, however, the monomers could not be delineated unambiguously, nor the TM segments assigned in a sequence-specific manner based on the experimental map alone.We have previously reported x-ray crystal structures of EmrE in the unbound form and in complex with TPP (15, 16). Regrettably, the electron density maps were calculated in the wrong hand because of an unfortunate change of sign of the anomalous differences (17), and helices were misassigned in the TPP-bound model. Recalculation of the...

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“…The addition of GamS protein helped preserve the PCR template so that sufficient levels of chaperone protein could be synthesized from the PCR amplified DNA templates. 12 This PCR DNA was still degraded by the end of the chaperone synthesis reaction as demonstrated by the lack of chaperone synthesis in the subsequent cell-free reactions (Fig. S1).…”

Section: Chaperone Sequential Expression Screenmentioning

confidence: 99%