Abstract
Ribonucleases are proteins whose use is promising in anticancer therapy. We have previously constructed different human pancreatic ribonuclease variants that are selectively cytotoxic for tumor cells by introducing a nuclear localization signal into their sequence. However, these modifications produced an important decrease in their stability compromising their behavior in vivo. Here, we show that we can significantly increase the thermal stability of these cytotoxic proteins by introducing additional disulfide bonds by site-directed mutagenesis. One of these variants increases its thermal stability by around 17 °C, without affecting its catalytic activity while maintaining the cytotoxic activity against tumor cells. We also show that the most stable variant is significantly more resistant to proteolysis when incubated with proteinase K or with human sera, suggesting that its half-live could be increased in vivo once administered.
Highlights
The increasing use of recombinant proteins for therapeutic purposes has highlighted certain issues, such as the control of the lifetime in the bloodstream or the long-term storage of the drug, that are related to the proteins’ stability
The stabilization should increase their resistance to proteolysis once they are distributed throughout the body
It has been described that the thermal stability of the antitumor RNases is an important factor in their cytotoxicity, likely by limiting their proteolytic degradation once the protein reaches the cytosol [1]
Summary
The increasing use of recombinant proteins for therapeutic purposes has highlighted certain issues, such as the control of the lifetime in the bloodstream or the long-term storage of the drug, that are related to the proteins’ stability. Maintenance of a protein drug’s native structure is critical for the specific interactions with the target molecules, and determines its solubility and its transport in a physiological environment. Along these lines, the production of new proteins with enhanced therapeutic abilities may be hampered by the reduction of the protein’s stability upon introduction of the designed mutations. Using strategies aimed to increase the stability of recombinant proteins is an important issue Among these strategies, we can mention the introduction of additional disulfide bonds [1], prolines [2], salt bridges [3], or the sequence modification to increase the propensity to adopt a given secondary structure [4]. RI is a protein that tightly binds to most pancreatic-type
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