Complementation of fragments

These observations on the complementation of fragments of nuclease are consistent with the concept that almost the entire amino acid sequence of nuclease is required for the minimum information necessary to determine a stable, functional nuclease structure. Furthermore, the native conformation of nuclease cannot be formed during assembly from the NH2-terminus until the polypeptide chain has been extended beyond residue 126 (80). Finally, it appears that stable and functional structures can be formed in several ways when the minimum requirement for information is fulfilled (81). 

Although slightly different from complementation of fragments, the ancient experiment of Fruchter and Crestfield (1965) on dimers of ribonuclease is the first example of in vitro complementation and must be cited to introduce this section. By complementation of ribonuclease inactivated by carboxy-methylation of either His 16 or His 119, but not both, formation of an active dimer was obtained in which 1 mole with free His 12 associates with 1 mole with free His 119. 

These examples indicate that a variety of combinations can give a functional structure. Other experiments describing complementation of fragments to yield nativelike structure have been reported. In most cases overall conformation of protein as examined by a physicochemical or an immunochemical probe was fully regained. Return of biological activity, while... 

The possibility of obtaining the values of association constants during the complementation of fragments, and also of studying the kinetics of the process is of great importance in the understanding of the conformational state of the individual fragments and of the structural adjustments required for their stabilization in the native structure. This aspect was particularly developed by Taniuchi and Bohnert (1975). 

IN VITRO COMPLEMENTATION OF FRAGMENTS CORRESPONDING TO STRUCTURAL DOMAINS... 

Fig. 10.9. Separation, purification, and complementation of fragments elastase T (16-125) and elastase T (126-245). Elution profile of proteins from a Sephadex G 100 column (a) N-acetyl [ C]elastase after limited proteolysis in a column equilibrated in 6 M GuHCl (100 mM acetate buffer, pH 5.0) (b) Purification of protein fragments (obtained as illustrated in a) by repeated chromatographic runs in the same conditions (c) Reassociation of fragments obtained in b. After incubation at pH 8, the proteins were fractionated in the column without GuHCl, at pH 8.0 (borate buffer, 50 mM), The arrows indicate the position corresponding to the elution of the intact protein (from Ghelis et al, 1978).

Mevel-Ninio and co-workers (1977) and Gervais and co-workers (1980) succeeded in splitting cytochrome 62 into different fragments, one with the heme group, the other with the flavin group, and to obtain reconstitution of the enzyme by complementation of fragments. 

Protein fragment complementation assays are based on an enzyme reassembly strategy whereby a protein-protein interaction promotes the efficient refolding and complementation of enzyme fragments to restore an active enzyme. The approach was initially developed using the reconstitution of ubiquitin as a sensor for protein-protein interactions (Johnsson and Varshavsky, 1994). Ubiquitin is a 76 amino acid protein that... 

Y-Specific sequences were enzymatically amplified from genomic DNA using the PCR.30 Two oligonucleotide primers, 21 bp in length, were synthesized. One of these matches a portion of the 5 end of the published 471-bp fragment from Sry. The second is the reverse complement of a... 

Complementing the structural studies of the intact transferrins, a number of fragments have also been crystallized, including proteolytic N-terminal half-molecules of rabbit serum transferrin (69) and chicken ovotransferrin (70), recombinant N-terminal half-molecules of human lactoferrin (71) and human serum transferrin (72), and a quarter-molecule fragment of duck ovotransferrin (73). All of these have now led to high-resolution structures (74-77). 

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