Protein expression systemic analysis

The analysis of libraries by flow cytometry requires special attention to be paid to the protein expression system. An ideal expression system for isolation of live cells by flow cytometry must fulfill the following criteria (Daugherty et al., 1999) ... 

Fig. 6.1 Comparative analysis of recombinant protein expression systems state-of-the-art versus magnICON . Background magnlCON -based expression of green fluorescent protein (GFP) in Ni-cotiana benthamiana (Plants are exposed to UV light). Left side insert Coomassie blue-stained polyacrylamide gel after SDS-electrophoretic sepa-...

A niunber of brain region proteomes have been studied to investigate differences in protein expression. Preliminary analysis of the mouse cerebellar proteome has identified 30 proteins (Beranova-Giorgianni, Giorgianni et al. 2002) and analysis of the porcine cerebellum led to identification of 56 spots (Friso and Wikstrom 1999). A developmental proteomic study of the rat cerebellum yielded resolution of over 3000 spots and identification of 67 of these (Taoka, Wakamiya et al. 2000). Most proteins showed an increase in abundance as the cerebellum matured, however, 42 spots appeared to be exclusively expressed in the immature cerebellum. Some of the latter were identified by MS and included proteins with defined roles in nervous system development. 

One of the best-studied carrier molecules is produced as a primary excretory constituent of the adult male mouse, known from its consistent high concentration as the major urinary protein (MUP). The basic 3-D structure of the protein was initially obtained from a monoclinic crystal of recombinant protein (MUP-I), constructed by induction in a bacterial expression system and purified to homogeneity (Kuser, 1990). A wild type version of MUP finally yielded to NMR analysis a clone of the r-isoform (162 residues) was labelled and compared with the crystal-structure (Lucke et al., 1990). Two views of the molecule... 

Membrane-integrated proteins were always hard to express in cell-based systems in sufficient quantity for structural analysis. In cell-free systems, they can be produced on a milligrams per milliliter scale, which, combined with labeling with stable isotopes, is also very amenable forNMR spectroscopy [157-161]. Possible applications of in vitro expression systems also include incorporation of selenomethionine (Se-Met) into proteins for multiwavelength anomalous diffraction phasing of protein crystal structures [162], Se-Met-containing proteins are usually toxic for cellular systems [163]. Consequently, rational design of more efficient biocatalysts is facilitated by quick access to structural information about the enzyme. 

Nunoi and co-workers (1988) fractionated neutrophil cytoplasm by Mono Q anion-exchange chromatography and obtained three fractions (NCF-1, -2 and -3) that were active in the assembly of the oxidase. Independently, Volpp and colleagues (Volpp, Nauseef Clark, 1988) prepared antiserum from cytosolic factors that eluted from a GTP-affinity column, and this antiserum (Bl) recognised cytoplasmic factors of relative molecular masses 47 kDa and 66 kDa. It was later shown by this group that these cytosolic factors translocated to the plasma membrane during activation. NCF-1 was shown to contain the 47-kDa protein and NCF-2 the 66-kDa protein. Analysis of the defect in the cytosol of autosomal recessive CGD patients revealed that most of these (88%) lacked the 47-kDa protein (p41 -phox), whereas the remainder lacked the 66-kDa protein (p66-phox). Both of these components have now been cloned and recombinant proteins expressed. Interestingly, in the cell-free system, recombinant p47-phox and p66-phox can restore oxidase activity of the cytosol from autosomal recessive CGD patients who lack these components. 

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