Regulation of recombinant proteína solubility and conformational quality in Escherichia coli

All the processes that take place in a cell require one or more proteins, meaning that they are essential components of life. Proteins are macromolecules consisting of amino acid units, all of them being constructed with combinations of only 20 amino acids. The primary structure of a protein molecule is determined by the sequence of amino acids connected by peptide bonds forming a polypeptide chain. Once the amino acid chain is synthesized, the protein folds by a physical process that might be eventually assisted by other proteins, reaching its characteristic three‐dimensional structure that is the final, functional conformation. However, although it is known that all the proteins must properly fold into their correct native conformation to be functional, their final conformation cannot be predicted from their primary amino acid sequence, being protein folding mechanisms one of the most challenging problems in biology today. Many proteins of relevant industrial or medical value are produced in low amounts in their natural sources. However, at the end of the seventies, the development of recombinant DNA technologies opened a new promising era for protein production in high amounts for both research and industrial applications. This had a tremendous impact, for example, in many areas of medicine as a tool to produce new drugs for the treatment of diseases and genetic disorders. Genetic engineering permits the introduction of the encoding genes of the protein of interest into recipient cells, where these genes are positioned downstream of regulable promoters in movable genetic elements, mainly plasmids. Under suitable conditions, these transgenic cells acting as protein production bio‐factories would be expected to act as unlimited and inexpensive source of rare, highly valuable proteins not only for proteomics and structural functional genomics1 but also for large‐scale preparative purposes. The quality as well as the quantity of the produced recombinant protein is greatly influenced by the chosen biological cell system. Bacteria have been the most commonly used organisms for protein production, specially the enterobacteria Escherichia coli, not only for the low cost of the used processes, but also for its fast growth. Generally, in Escherichia coli, the rather small host cell proteins can fold properly, adopting a native, biological active conformation. However, when producing heterologous proteins, specially those with eukaryotic or viral origin, important obstacles appear during the protein production process: a) in most cases, the protein is produced in a non functional conformation; b) sometimes the formed product is toxic for the cell; c) the protein often results proteolytically degraded2; d) the product is accumulated as an insoluble, non‐functional protein aggregates, known as inclusion bodies3. Therefore, even though the important advantages of the use of bacteria as a expression system, Escherichia coli also presents some drawbacks, such as its inability to carry most of the post‐transcriptional modifications, often required for eukaryotic protein function, the lack of a secretion mechanism to release the protein to the medium, and the inability to create an oxidative environment to facilitate disulfide bond formation required to achieve the final, functional structure of some proteins. Therefore, this leads to the production of proteins which are not always suitable for immediate use. This means that, to date, many proteins have been excluded from the biotechnological and pharmaceutical market because they cannot be produced in high yields as soluble and active products. To avoid protein folding problems encountered in bacteria under overexpression conditions, mainly secretion and post‐transcriptional modifications, alternative host cells, such as yeast, filamentous fungi, mammalian or insect cells, have been explored. Nevertheless, an enormous number of deficiencies in these systems such as difficulty of genetic manipulation, low productivity and high costs, shows that these organisms are not ideal for this aim and that, even when bacteria show some obstacles in the production process and often this system has to be optimized for specific products, it is, in most of the cases, the best choice