Expression vectors, fluorescent proteins

A large number of different phage display vectors have been constructed. With pretending to be complete, Table 1 lists a selection of phage display vectors. Some of them have not been used for the construction of a library up to now but have been included because they offer possible alternatives. For example, one of the systems allows the success of antibody gene cloning to be monitored by the expression of green fluorescent protein (52). 

In a project implemented at Nil, ovine growth hormone (oGH) was expressed as inclusion bodies using a pQE expression vector. The expressed protein was 10 -15% of the total cellular proteins of E. coli. The recombinant ovine growth hormone was purified from inclusion bodies and refolded into its native conformation. The refolded recombinant oGH was found to be immunoreactive with RIA/RRA characterized by SDS-PAGE, Western blot, N-terminal amino acid sequence, CD, fluorescence spectra, RIA, and RRA. High cell density cultivation was standardized and found to be enhanced to 100 times. In 16 h, 3.2 g H of oGH was produced which is claimed to be the highest level of any recombinant growth hormone reported in E. coli [57]. 

Expression Vectors for Fluoresc it Proteins Several plasmids with which to fuse the chemokine receptor to fluorescent proteins are commercially available. Some allow inclusion of the fluorochrome at the N-terminal end of the receptor and others at the C-terminal end (e.g., pEYFP-Cl, pEYFP-Nl or pDsRed-Monomer N1 vectors from Clontech, Mountain View, CA). 

For rapid, easy visualization, one can use fluorescent protein-based constructs and microscopy techniques. Such constructs are typically employed in colocalization analysis and energy-transfer techniques. Receptors are cloned using standard molecular biology techniques into commercially available vectors bearing fluorescent proteins. Insertion of the fluorescent probe in the C-terminal region of the receptor involves elimination of the receptor stop codon, whereas insertion in the N-terminal region requires elimination of the fluorescent protein stop codon. Transfected cells should be analyzed for receptor expression and ftmction (reeNote 8). 

The construction of fluorescendy labeled receptors uses standard molecular biology techniques. The fluorescent probe must be inserted in the C-terminal region to conserve the original signal peptide of the receptor. The fusion involves elimination of the receptor stop codon and insertion of the receptor into the fluorescent protein vector, separated by several additional amino acids. Transfect cells and analyze for correct expression and function. 

For the integrating vector constructs we are using to express fluorescent proteins under control of the actin 15 promoter, there are two ways of modulating the expression level. First, to select an appropriate clone after transformation of the cells (for details see(6)). These vectors integrate randomly into the genome, and the level of expression depends on the site of integration. Second, even in a clonal population the expression varies from cell to cell. If green and red fluorescent proteins are expressed simultaneously, their levels fluctuate independently. Therefore, the experimentalist has to choose cells within a population that exhibit the optimal levels of expression (tee Note 4). 

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