Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes.

In this paper, the successful expression of trypanosomal triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Escherichia coli, using a "7-polymerase-based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure.A TIM mutant was made in which a unique dimer-interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could be expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wildtype TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild-type trypanosoma1 TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution-dependent inactivation is reversible. His47 is involved in a water-mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water-mediated hydrogen bond is important for the stability of the TIM dimer.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.

It is often assumed that there is sufficient information contained within the primary amino acid sequence of proteins for the correct three dimensional structure to be attained. However, for many proteolytic enzymes it has been shown that the presence of pro-regions is essential for correct folding to occur. The pro-regions also act as a control mechanism to prevent uncontrolled proteol ysis.