Phase separation protein

Figure 9.4. (A) Transformed E. coli with more than 70% of the cell volume filled by huge inclusion bodies of the phase separated protein-based polymer (GVGVP)i2i. (B) Nontrans-formed E. coli without product. (Reproduced with permission from Urry et al. )...

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

Aqueous-detergent solutions of appropriate concentration and temperature can phase separate to form two phases, one rich in detergents, possibly in the form of micelles, and the other depleted of the detergent (Piyde and Phillips, op. cit.). Proteins distribute between the two phases, hydrophobic (e.g., membrane) proteins reporting to the detergent-rich phase and hydrophilic proteins to the detergent-free phase. Indications are that the size-exclusion properties of these systems can also be exploited for viral separations. These systems would be handled in the same way as the aqueous two-phase systems. 

Phase separation to remove ELP-DNA binding protein Plasmid elution from ELP-DNA binding protein... 

It has been shown by FM that the phase state of the lipid exerted a marked influence on S-layer protein crystallization [138]. When the l,2-dimyristoyl-OT-glycero-3-phospho-ethanolamine (DMPE) surface monolayer was in the phase-separated state between hquid-expanded and ordered, liquid-condensed phase, the S-layer protein of B. coagulans E38/vl was preferentially adsorbed at the boundary line between the two coexisting phases. The adsorption was dominated by hydrophobic and van der Waals interactions. The two-dimensional crystallization proceeded predominately underneath the liquid-condensed phase. Crystal growth was much slower under the liquid-expanded monolayer, and the entire interface was overgrown only after prolonged protein incubation. 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

Tzannis and Prestrelski [144,145] showed a concentration-dependent protective effect of sucrose and residual moisture on the activity and stability of tryp-sinogen during spray-drying. They found protein protection even at low carbohydrate concentrations, but they observed some destabilization at very high sucrose concentrations. They hypothesized that phase separation occurs at high sucrose concentrations, resulting in... 

T. W. Randolph, Phase separation of excipients during lyophilization Effects on protein stability, J. Pharm. Sci., 86(11), 1198 (1997). 

Wall, D. B. Kachman, M. T. Gong, S. Hinderer, R. Paras, S. Misek, D. E. Hanash, S. M. Lubman, D. M. Isoelectric focusing nonporous RP HPLC A two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. Anal. Chem. 2000, 72, 1099-1111. 

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 application of polymer monoliths in 2D separations, however, is very attractive in that polymer-based packing materials can provide a high performance, chemically stable stationary phase, and better recovery of biological molecules, namely proteins and peptides, even in comparison with C18 phases on silica particles with wide mesopores (Tanaka et al., 1990). Microchip fabrication for 2D HPLC has been disclosed in a recent patent, based on polymer monoliths (Corso et al., 2003). This separation system consists of stacked separation blocks, namely, the first block for ion exchange (strong cation exchange) and the second block for reversed-phase separation. This layered separation chip device also contains an electrospray interface microfabricated on chip (a polymer monolith/... 

These columns have been used for separation of proteins of over 200 kDa MW in our experiments as shown by analysis using a ID gel. In addition, columns with larger particle sizes have been used to separate proteins of over 400 kDa (55-56). The NPS RP-HPLC method provides a liquid phase method for separating large intact proteins for further analysis. More specifically, it provides a means of separating proteins for interfacing to mass spectrometric analysis. 

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