Using four different Pichia Pink

Using four different Pichia Pink strains and two different pPink vectors (Table 1), eight different yeast strains were obtained. The level of recombinant proteins produced was compared in the eight strains by P4H radioactive enzymatic assay. The highest enzymatic activity was obtained in the αβH4 strain cell lysate obtained by transformation of a P. pink strain defective for two different vacuolar proteases with the High-copy pPink vector (Fig. 2A). The analysis of the inserted gene copy number revealed that in this case there was no correlation between the P4H enzymatic activity and αP4H inserted gene copy number (Fig. 1A). In contrast, an inverse correlation between yeast intracellular protease activity and the recombinant P4H enzyme stability could be observed by comparing the enzymatic activity of P4H in the various protease-defective yeast strains (Fig. 2A) especially in the High-copy transfected strains (H strains). In fact, strain H1, with the lowest recombinant activity, has a completely efficient protease apparatus, strain H2 and H3, both measuring half recombinant enzymatic activity respect to strain H4, are defective for just one protease while strain H4, the best performing, is the one lacking most proteases (proteinases A and B, that consequently can neither activate carboxypeptidase Y), a condition clearly increasing the survival of the recombinant proteins inside the yeast NVP-LCQ195 receptor (Cregg et al., 2000). Surprisingly, an enzymatic activity in the P. pastoris strain transformed only with the α subunit was also measured, indicating that sponge αP4H, differently from its human counterpart (Vuorela et al., 1997), could at least partially assemble with the yeast PDI, or alternatively the sponge enzyme could partially hydroxylate without the support of its β subunit, with a measured enzymatic activity 46.2±3.6% lower than the activity of the whole α and β P4H sponge tetramer (data not shown). The enzymatic activity of recombinant sponge P4H was assayed in vitro both at 37°C, the standard enzymatic kinetics temperature, and at 15°C, the typical sea temperature of C. reniformis marine environment (Fig. 2C). Surprisingly the data obtained indicate that the sponge enzyme has a lower activity at its natural environment temperature. As a matter of fact, the proline hydroxylation rate on collagen chains is minimal in the heterothermal animals living in cold marine environments, and conversely its hydroxylation is enhanced in animals living in warmer seas to increase collagen thermal stability (Kimura et al., 1988). Thus, the behavior of the recombinant sponge P4H suggests that the enzyme can be able to modulate its activity allowing the animal to respond to sea temperature alterations by adjusting the levels of collagen hydroxylation to the necessities of the environmental changes. Western blot analysis of αβH4 strain lysate after methanol induction could confirm that also in the recombinant yeast system, α and β polypeptides are assembled as tetramers (Fig. 3B) as already observed for the wild type protein analyzed in C. reniformis cell extracts (Pozzolini et al., 2015). Finally, the enzymatically best performing strain αβH4 was transformed with the expression vector pPICZ\\colCH containing a C. reniformis non fibrillar collagen gene, producing the colCH4 strain. This strain shows an enhanced recombinant P4H enzymatic activity with respect to the αβH4 lacking the collagenous sequence (Fig. 4D). These data seem to confirm results obtained by others (Vuorela et al., 1997) the explanation of which was ascribed at a higher stability of the P4H tetramer in the presence of its natural substrate in the recombinant system. But surprisingly, when we analyzed the levels of each recombinant transcript in our system we found that, differently from previous work, the enhanced enzymatic activity seems to be mainly linked to an enhanced transcription of the α and β P4H recombinant genes in the presence of the ColCH gene as displayed in Fig. 4A and B. Indeed, it has been reported that genes with promoters susceptible of activation by the same transcription factors tend to localize in close proximity in the nucleoplasmic space likely in the same interchromatin compartment to be optimally transcribed by a single transcription factory (Larkin et al., 2013). Thus, in this case the introduction of further copies of another recombinant gene (ColCH) in the αβH4 strain may have the effect to promote a more efficient transcription also of the other two inserted genes (α and β P4H) localized on different chromosomes but sharing the same promoter sequence. This phenomenon, which surely needs further investigation, could be of importance when setting up the production of multiple recombinant proteins in yeast since, surprisingly, it seems to suggest that the more proteins are introduced under the same promoter the more efficient becomes their production.