Protein Splicing: From the Foundations to the Development of Biotechnological Applications.

Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size. This methodology has been developed based on the knowledge obtained from protein splicing. Protein splicing is a multistep biochemical reaction that includes the concomitant cleavage and formation of peptide bonds carried out by self-processing domains named inteins. The natural substrates of protein splicing are essential proteins found in intein-containing organisms; inteins are also functional in nonnative frameworks and can be used to alter nearly any protein's primary amino acid sequence. Accordingly, different reactivity features of inteins have been largely exploited to manipulate proteins in countless methods encompassing fields from biochemical research to the development of biotechnological applications including the study of disease progression and validation of potential drug candidates. Here, we review almost three decades of research to uncover the chemical and biochemical enigmas of protein splicing and the development of inteins as potent protein engineering tools.

Construction of Highly Stable Cytotoxic Nuclear-Directed Ribonucleases.

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.