Renaturation factors

Muller, C. and Rinas, U., Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography, /. Chromatogr. A, 855, 203, 1999. 

Stem, B. D., Wilson, M., and Jagus, R. (1993). Use of nonreducing SDS-PAGE for monitoring renaturation of recombinant protein synthesis initiation factor, eIF-4 alpha. Protein Expr. Purif. 4, 320-327. 

The energy dependence of import into mitochondria has been exploited to accumulate large amounts of precursors in an uncoupler-poisoned living cell [93]. Precursor to the 8-subunit of the proton ATPase has been purified from such cells by affinity chromatography on an antibody column, followed by chromatofocussing and isoelectric focussing. After renaturation, this precursor can be correctly processed by the matrix protease and can be imported into mitochondria, but only in the presence of a proteinaceous factor from the yeast cytoplasm [94]. A similar finding has been reported for import of precursors into rat liver mitochondria in which a factor is provided by the reticulocyte lysate [95,96]. 

Calf thymus DNA, Xenopus liver DNA, and also DNA from T4 coli phage inhibit the vegetalizing factor. Heteropolymeric [poly d( A-T), poly d(G-C), poly d(I-C)] and homopolymeric (poly I-poly C, poly dA poly dT) double-stranded polynucleotides have no inhibitory activity (Table I). Melted, single-stranded chick DNA has about the same inhibitory effect as double-stranded DNA. However, it is not excluded that a partial renaturation occurs under the conditions of the test. Also, DNA, which was partially degraded by sonication, has about the same inhibitory effect as untreated DNA. 

The stability of a DNA duplex depends on, besides the chemical and physical structure of the DNA itself, many environmental factors such as solvent, temperature, and salt composition and concentration. For example, when a stable DNA duplex is heated to higher temperatures, the double helix structure will be gradually denatured into two coiled single strands. When cooled to room temperature, the two single strands can reassociate to again form the duplex, a process called DNA renaturation (sometimes also called DNA hybridization or annealing). These processes are reversible, as illustrated in the following equilibrium ... 

The most convenient and comparable measurement of enzyme stabUity is the half-life at a given temperature (other incubation conditions being defined), i.e. the time period over which half the initial activity of an enzyme is lost. Additional information on the effect of temperature on activity can be important, for instance the shape of a plot of logio of percent activity remaining versus time of incubation can indicate whether irreversible thermal denaturation is the sole eause of loss of activity or whether additional factors are involved, such as autolysis, renaturation, or substrate effects. 

As considered in the following section, several factors influence the kinetics of denaturation and renaturation. A thorough understanding of the denaturation/ renaturation phenomenon as well as the factors influencing the process should prove valuable in performing and refining the many important techniques mentioned earlier. 

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