By Henry Tedeschi
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Extra resources for Cell physiology: molecular dynamics
2001). GFP-cDNA attached to a promoter provides a useful reporter gene. Chimeric DNA coding for a protein and GFP serves as a useful marker to trace the fate of newly synthesized proteins. , 1997). The major disadvantage of the approach is that GFP can interfere with the function of its fusion partner (see for example Doyle and Bolstein, 1996, showing that actin-GFP fails to function appropriately). , 2001) GFP-fusion proteins containing targeting information (in the form of an amino acid domain such as a signal sequence) can be produced by manipulation of cDNA.
Sequencing of Proteins and Computer Analysis Techniques are available for sequencing proteins; in fact, the methodology has been automated. In practice it is easier to sequence DNA because of the capacity to multiply the DNA either through cloning or PCR, so that the amino acid sequence is most frequently deduced from the nucleotide sequence. The methodology has been standardized to the extent that it is possible to commercially contract for deriving sequencing information from purified compounds.
Proteins that specifically bind to other proteins can be identified by an ingenuous approach referred to as interaction cloning (Blanar and Rutter, 1992). With this technique, the cDNA of a known protein is modified by adding the coding sequence for the phosphorylation site of heart muscle kinase. The composite protein is then expressed in E. Coli and after purification and labelling with [32P], it serves as a probe for a λgt11 cDNA expression library from eukaryotic cells under study. coli plaques containing the unknown protein binding the radioactive probe can then be detected and the λgt11-cDNA isolated and cloned (see Section IIA).