Heterologous and Homologous Expression of Proteins from Haloarchaea: Denitrification as Case of Study

Haloarchaea (halophilic microbes belonging to the Archaea domain) are microorganisms requiring mid or even high salt concentrations to be alive. The molecular machinery of these organisms is adapted to such conditions, which are stressful for most life forms. Among their molecular adaptations, halophilic proteins are characterized by their high content of acidic amino acids (Aspartate (Asp) and glumate (Glu)), being only stable in solutions containing high salt concentration (between 1 and 4 M total salt concentration). Recent knowledge about haloarchaeal peptides, proteins, and enzymes have revealed that many haloarchaeal species produce proteins of interest due to their potential applications in biotechnology-based industries. Although proteins of interest are usually overproduced in recombinant prokaryotic or eukaryotic expression systems, these procedures do not accurately work for halophilic proteins, mainly if such proteins contain metallocofactors in their structures. This work summarizes the main challenges of heterologous and homologous expression of enzymes from haloarchaea, paying special attention to the metalloenzymes involved in the pathway of denitrification (anaerobic reduction of nitrate to dinitrogen), a pathway with significant implications in wastewater treatment, climate change, and biosensor design.

A Brief Reminder of Systems of Production and Chromatography-Based Recovery of Recombinant Protein Biopharmaceuticals

Recombinant proteins are produced for various applications in laboratory and industrial settings. Among them, therapeutic applications have evolved into a mature field in recent years, affecting the face of contemporary medical treatment. This, in turn, has stimulated an ever-greater need for innovative technologies for the description, expression, and purification of recombinant protein biopharmaceuticals. Therefore, many biopharmaceuticals are synthesized in heterologous systems to obtain satisfactory yields that cannot be provided by natural sources. As more than 35 years has passed since the first recombinant biopharmaceutical (human insulin) successfully completed clinical trials in humans, we provide a brief review of the available prokaryotic and eukaryotic expression systems, listing the advantages and disadvantages of their use. Some examples of therapeutic proteins expressed in heterologous hosts are also provided. Moreover, technologies for the universal extraction of protein molecules are mentioned here, as is the methodology of their purification.

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

ABSTRACTThe gene mutated in Huntington’s disease (HD) patients encodes the 348 kDa huntingtin (HTT) protein. The pathogenic HD CAG-expansion mutation causes a polyglutamine (polyQ) tract at the N-terminus of the HTT protein to expand above a critical threshold of ~35 glutamine residues. The effect of HD mutations on HTT is not well understood, in part due to difficulties in carrying out biochemical, biophysical and structural studies of this large protein. To facilitate such studies, we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprises both N and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, juvenile HD patients as well as the more extreme polyQ expansions used in some HD tissue and animal models. These reagents yield milligram quantities of pure recombinant HTT protein, including many of the previously mapped posttranslational modifications. We have characterised both apo and HTT-HAP40 complex samples produced using this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful complexes of HTT. We demonstrate how these resources can produce sufficient material for protein-intensive experiments such as small angle X-ray scattering (SAXS), providing biochemical insight into HTT protein structure. The work outlined in this manuscript and the tools generated, lay a foundation for further biochemical and structural work on the HTT protein and its functional interactions with other biomolecules.