Staphylococcal nuclease isolation

Figure 7 Experimental and theoretical inelastic neutron scattering spectrum from staphylococcal nuclease at 25 K. The experimental spectrum was obtained on the TFXA spectrometer at Oxford. The calculated spectrum was obtained from a normal mode analysis of the isolated molecule. (From Ref. 28.)...

Staphylococcal nuclease, a DNA-hydrolyzing enzyme, is a single polypeptide chain of 149 amino acid residues it has no disulfide bridge and contains one Ca ion. This protein was originally isolated from Staphylococcus aureus, but the gene has been cloned and inserted into several expression systems. Various mutants of this enzyme have been used to generate biochemical and biophysical data on the structural and functional consequences of altering the protein amino acid sequence. 

Staphylococcal nuclease was initially isolated as an extracellular enzyme (nuclease A) of S. aureus (or Micrococcus pyogenes) (1,49). The extremely high affinity of the nuclease to phosphocellulose at pH 5.8 has provided the basis of convenient purification methods. SNase can also be efficiently purified by the use, as an affinity matrix, of 3 -(4-aminophenylphosphoryl)deoxythymidine 5 -P linked to the CNBr-activated Sepharose (50). 

An immunochemical approach was used to study conformational equilibria of isolated polypeptide fragments as well as entire staphylococcal nuclease (Sachs et al, 1974 Furie et al, 1975). An antibody population directed against the unfolded form of fragments (99-126)r was isolated to study the equilibrium between unfolded nuclease and the spontaneously refolded form. Figure 9.8 is a schematic representation of the presumed two-state equilibrium between native and unfolded forms. It may be described by the following expression ... 

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