Proteins vectors

Figure 6.27 Representation in the Gaussian plane of the phase relationships derived by single isomorphous replacement, SIR, in a protein. The structure factor of the protein vector, FP, lies on a circle of radius FP centred at O. The structure factor of the heavy metal derivative, FPH, lies on a circle of radius Fph, with centre at the tip of the vector -Fh- The intersection of the two circles represents the two solutions to equation (6.6). The resulting vector FP can be drawn in two positions, corresponding to two different phase angles...

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. 

The dot blot will provide a qualitative estimate of antibody titer (Protocol 21.3). The same test can be done quantitatively by ELISA (enzyme-linked immunosorbent assay), but the technically simpler dot blot gives adequate information, uses less protein, and does not require an ELISA plate reader. If the same protein has been expressed in two different fusion-protein vectors, the test should be performed with the protein not used for immunization. If there is only one fusion protein, e.g., a GST-fusion, then a second dot blot can be done in parallel with just GST alone to compare the titers. If the antiserum recognizes the protein, there should be a lower titer of antibodies against GST alone as compared to against the entire fusion protein. A preimmune control is always advisable. Figure 21.1 shows a sample dot blot from a monoclonal antibody screen. 

The forces in a protein molecule are modeled by the gradient of the potential energy V(s, x) in dependence on a vector s encoding the amino acid sequence of the molecule and a vector x containing the Cartesian coordinates of all essential atoms of a molecule. In an equilibrium state x, the forces (s, x) vanish, so x is stationary and for stability reasons we must have a local minimizer. The most stable equilibrium state of a molecule is usually the... 

If an antibody to the protein of interest is available, it is sometimes possible to use vector sequences, eg, the beta-galactosidase promoter sequence, to direct the transcription of the passenger DNA into messenger RNA and the translation of that mRNA into protein which can be recognized by the antibody. Although this method is somewhat less reHable than the use of nucleic acid probes, specialized vectors are available for this purpose. 

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

How do we find phase differences between diffracted spots from intensity changes following heavy-metal substitution We first use the intensity differences to deduce the positions of the heavy atoms in the crystal unit cell. Fourier summations of these intensity differences give maps of the vectors between the heavy atoms, the so-called Patterson maps (Figure 18.9). From these vector maps it is relatively easy to deduce the atomic arrangement of the heavy atoms, so long as there are not too many of them. From the positions of the heavy metals in the unit cell, one can calculate the amplitudes and phases of their contribution to the diffracted beams of the protein crystals containing heavy metals. 

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

FIGURE 13.17 A protein expression vector contains the hybrid promoter derived from fusion of the lac and trp promoters. Expression from pi A more than 10 times greater than expression from either the lac or trp promoter alone. Isopropyl-/3-D-thiogalacto-side, or IPTG, induces expression from pi as well as lac. 

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