E. coli lysate

Cargile, B. J. McLuckey, S. A. Stephenson, J. L. Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions. Anal. Chem. 2001, 73,1277-1285. 

Nelson, R. W. Jarvik, J. W. Taillon, B. E. Tubbs, K. A. BIA/MS of epitope-tagged peptides directly from E. coli lysate Multiplex detection and protein identificatin at low-femtomole to subfemtomole levels. Anal. Chem. 1999, 71, 2858-2865. 

CEC RPLC UV, MS Protein mix and E.coli lysate 8-port Opiteck et al. (1997)... 

FIGURE 8.8 2D chromatogram of a SEC x RPLC separation of a native E. coli lysate. Reprinted from Opiteck et al. (1998), by permission of Academic Press. 

FIGURE 8.12 UV absorbance chromatograms of three anion-exchange separations of an E. coli lysate. Vertical lines represent the times at which fractions were changed. 

TABLE 8.2 Summary of the Data from Three LC x UHPLC Separations of an E. coli Lysate... 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

Results from a Western blot. A SDS-PAGE gels, 12%, were run and transferred to nitrocellulose Lane 1, MW standards lane 2, biotinylated standards lane 3, human transferrin, lane 4, E. coli lysate, lane 5, total human serum, lane 6, biotinylated standards Gel A was stained with a protein dye Blot B was assayed using rabbit anti-human transferrin as the first antibody The second antibody solution contained anti-rabbit HRP conjugates Only the transferrin bands and the prestained biotinylated standards were detected by the antibodies and the avidin-HRP treatment... 

The lysate was obtained from E. coli cells which were pre-transformed with a plasmid (pSTB7) expressing tryptophan synthase from Salmonella enterica. This E. coli strain is commercially available (ATCC 37845). Reported yields on tryptophans ranged from 3% to 63%, but, in several cases, could be significantly enhanced up to 100% using the E. coli lysate, separated from the reactant by a dialysis tube. 

Inserted tip PC chip 200 nL/min ESI-ITMS BSA (30 jxM), cytochrome c (8 jxM), ubiquitin (2.4 jxM), E. coli lysate Flat-Edge ESI Sprayer Dialysis Nil 815... 

Figure 2. Comparison of blank run reference impurities obtained from a single E. coli lysate but purified through the process in the presence of growth hormone (panel A, arrow) or the absence of growth hormone (panel B). These silver stained 2-D gels demonstrate that the absence of the product did not significantly change the distribution of impurities and therefore their chromatographic behavior in the process.

Recombinant wild type hIL-ip, the K138C mutant and the K138C, R4A, L6A triple mutant (mutant 1) were isolated from the soluble fraction of E. coli lysates by ammonium sulfate fractionation and hydrophobic interaction chromatography. The purified proteins were characterized by SDS-PAGE, western blots, N-terminal sequence, size exclusion chromatography (SEC), isoelectric focusing (lEF), matrix assisted laser desorption ionization mass spectrometry (MALDI-MS), and electrospray mass spectrometry (ESMS). 

Recombinant uridine phosphorylase from E. coli lysate [9]... 

The capability of the XLL cross-axis CPC was further examined in the purification of a recombinant UrdPase from a crude E. coli lysate under the same experimental conditions as described earlier. The majority of the protein mass was eluted immediately after the solvent front (between 105 mL and 165 mL elution volume), whereas the enzyme activity of UrdPase coincided with the fourth... 

Figure 6. Flow decay curve for concentrating E. coli lysate with a 0.45 pm microporous (Durapore) membrane. Inlet pressure was 90 psi outlet pressure, 30 psi. The initial volume was 6.8 liters the final volume, 1.3 liters.

Pyruvate dehydrogenase and pyruvate-formate lyase are enzymes that are endogenous to E. coli and which process pyruvate into acetyl coenzyme A. Acetyl phosphate is then produced from acetyl coenzyme A by phosphotransacetylase, also endogenous to E. coli. In this way, pyruvate can be processed in E. coli to yield acetyl phosphate, a phosphate donor (3). The activities of these pathways in E. coli lysate were tested [7] by supplying a batch-mode reaction with pyruvate in the absence of an alternative secondary energy store. 

Fig. 10.3-16(d). After the labeling step, the protein was attached to avidin coated glass for further screening applications. The use of an exogenous transferase for the labeling step is advantageous, as the native transferases present in the E. coli lysate do not recognize the protein domain or substrate 44. 

---