Membrane separations, lysate

Figure 4. Purification of PemB from E. coli K38 pGPl-2/pPME6-5 cells. Proteins were separated by urea-SDS-PAGE. Lane 1, induced cell lysate lane 2, soluble protein fraction from induced cells lane 3, membrane fraction from non-induced cells lane 4, membrane fraction from induced cells lane 5, membrane proteins not extracted by Triton X-100 lane 6, membrane proteins extracted by Triton X-100 lane 7, PemB purified by preparative electrophoresis. The molecular weight standard positions are indicated.

Zhu, Y. Lubman, D. M. Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations. Electrophoresis 2004, 25, 949-958. 

The cells and cell lysates (fragments of disrupted cells) can be separated from the soluble components by using microfiltration (Chapter 8) with membranes. This separation method offers following advantages ... 

A recent development is to combine filtration with solid-phase extraction separation. These filter modules contain a unique silica gel membrane that binds up to 20 pg of DNA in the presence of a high concentration of chao-tropic salt and allow eventual elution in a small volume of low-salt buffer. They also contain an asymmetric laminar membrane with a gradation of pore sizes for efficient removal of material precipitated in the lysate. Such membrane filters eliminate time-consuming phenol-chloroform extraction and alcohol precipitation. The impregnation of silica in the membrane matrix also prevents the problems associated with loose resins and slurries. High-purity plasmid DNA eluted from such modules is ready to use and often needs no further precipitation, concentration, or desalting. 

To answer the above issues, a model system was devised in which a specific protein was added to ah IS. coll lysate. The lysate was then processed through the filtration steps and the protein recovered. The protein chosen was human IgG which has a relatively large molecular weight (160,000 Daltons). It was reasoned that if a large protein could be separated from cell debris and passed through a membrane and recovered, then a smaller protein that is typical of a recombinant process, should prove to be much easier. 

The separation,purification and concentration of a thermosensitive bioactive compound from a lysate has been carried out combining UF, ion exchange and RO with significant cost reduction and productivity increase. Enzyme membrane reactors have been used for triglyceride enzymatic hydrolysis and product separation. Thermophi1ic,thermostable enzyme ultrafiltration membrane have been prepared, and used in high temperature lactose hydrolysis. 

Quantitative protein analysis is accomplished by combining protein separation, most commonly by high-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE), with MS-(mass spectrometry) based or tandem MS (MSZMS)-based sequence identification of selected, separated protein species. This method is sequential, labor intensive and difficult to automate. It selects against specific classes of proteins, such as membrane proteins, very lar ge and small proteins, and extremely acidic or basic proteins. Tlie technique has a bias toward highly abundant proteins, as lower abundance regulatory proteins (e.g., transcription factors and protein kinases) are rarely detected when total-cell lysates are analyzed. 

Transcription, translation, and import reactions. The procedures used have been previously described (1). A wheat gene (9) was inserted 3 to the SP6 promoter and the transcripts produced using SP6 polymerase were translated in either a wheat germ or reticulocyte lysate. After import reactions, the pea chloroplasts were either left untreated and hypotonically lysed, or treated with thermolysin to remove proteins on the exterior. Membrane and soluble fractions were separated by centrifugation and analyzed by SDS-PAGE. 

Quake and coworkers [16] developed a PDMS microfluidic device (shown in Eig. 4c) for nucleic acid purification from a small number of bacterial or mammalian cells. This multilayer device contained fluidic channels and a system of membrane-actuated pneumatic valves and pumps, which enabled precise control of buffers, lysis agents and cell solution, and also allowed for parallel processing. Bacterial cells, dilution buffer and lysis buffer are first introduced into the chip and then transferred into the rotary mixer. Once mixed, the lysate is flushed over a DNA affinity column and drained. The DNA is recovered from the chip with an elution buffer for further analysis. We note that this is the only microfluidic chemical C3flome-try device to use a separation method other than solution-phase electrophoresis (i. e., solid phase extraction). 

Intracellular drug concentrations of L1210 cells incubated with [3r]MTX, [ H]MTX-BSA and [ H]MTX-Dextran T70 at 0 and 37 . 2 x 10 cells were incubated for 1 hr with either 4 x 10 M drug alone or with the addition of the transport inhibitor or competitor indicated. After lysis, lysate and membrane fractions were separated by centrifugation and the lysate dialyzed against water. 

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