Disulfide-bonded proteins, synthesis

In the broadest terms, cell-free synthesis offers advantages over traditional in vivo methods of recombinant protein expression, as it enables the experimentalist strictly to control conditions under which expression reactions are performed. Benefits of such an open expression system are exemplified by the fact that cell-free reaction conditions can be sufficiently modified to support co-translational folding of active disulfide-bonded proteins. 

Treatment of Extracts for Synthesis of Disulfide-bonded Proteins... 

Protein synthesis and folding Signal peptide cleavage Formation of disulfide bonds Oligomerization A/-Clycosylation folding... 

Ribonuclease A (RNase A) was selected as the target enzyme for solid-phase synthesis because its sequence was known (Scheme S), 22 25 and an X-ray structure had been deduced. 24 Importantly, it had been shown that this 124-residue protein could be reduced and unfolded and then reoxidized to re-form the four disulfide bonds with recovery of full enzymatic activity. 25 ... 

The new solvent systems (Section 5.1.7.1) were an important advance and opened the way for the solution segment synthesis of even larger and more complex proteins. For example, human midkine (121 residues) containing 5 disulfide bonds was assembled from Boc-(l -59)-OH and H-(60-121)-OBzl, which were not soluble in DMSO, but were readily soluble in CHCl/TFE (3 1) (Scheme 12).[441 The coupling was undertaken with a water-soluble car-bodiimide containing HOOBt and was complete in one hour. 

Finally, some authors propose the use of enzyme mixtures for the biosynthesis of complex molecules. A cell free protein synthesis (CFPS) system is a novel approach that was successfully used for the production of complex mammalian proteins with multiple disulfide bonds (Bhatta-charya, 2004). However, even if the technical potential of this method is proved, its economical feasibility has still to be proven. 

Examination of genotoxicity of pharmaceuticals is required to assess the interaction of the drug with DNA. These studies are generally not applicable to immunotoxins. Unlike chemotherapeutics that cause cell death through DNA interaction, immunotoxins mediate cell death by preventing protein synthesis. However, immunotoxins use a linker to connect the toxin to the antibody that may need to be examined if it is an organic linker and has the ability to bind DNA (per ICH S6). The majority of immunotoxins use either a nonreducible thioether linker for intact toxins or a disulfide bond for A chains and ribosome-inactivating proteins and do not interact with DNA. 

Clearly, the action of prolyl isomerases is not restricted to the slow folding of polypeptide chains with intact disulfides, but they also accelerate the oxidative folding of reduced proteins, which resemble more closely the nascent polypeptide chains as they occur in the endoplasmic reticulum. The simultaneous presence of PPI markedly enhances the efficiency of PDI as a catalyst of disulfide bond formation. Both enzymes act according to their specificity and catalyze the isomerization of prolyl peptide bonds and the formation of disulfide bonds, respectively, in the folding protein chains. It remains to be demonstrated that a similar concerted action of the two enzymes can take place in the course of de novo synthesis and folding of proteins in the cell. 

---