Protein Production


Since the 1970s, the production of recombinant proteins has become a driving force in the study of new drugs and vaccines and the implementation of new biotechnological and biopharmaceutical applications. Indeed, the pharmaceutical industry has demanded an increasing variety of recombinant proteins in order to develop new bio-strategies to address the impact of various diseases on human life. To overcome this situation, recently, although there is no single perfect host for every protein, a large number of proteins have been expressed in different expression systems, ranging from the bacterial host, such as Escherichia coli, to yeast and mammalian cells, with high throughput due to the rapid development of genetic engineering technologies. Although Escherichia coli does not ensure post-translational modifications and many human proteins have been expressed at a low level due to their incompatibility with the prokaryotic system, Escherichia coli remains the most widely used host due to its manageability, low cost, and high protein yield. The main goal of protein production is to obtain a substantial amount of pure, folded protein. The laboratory-scale procedure for recombinant protein production in E. coli consists of consecutive steps, starting from an initial theoretical planning phase to a practical phase in which cells are transformed, grown, and obtained overexpression of the protein of interest. In the first phase, certain strategies, such as host type, plasmid type, cell strain, culture parameters for protein expression, and choice of culture medium (unlabeled and labeled), must be defined in order to optimize, as much as possible, the subsequent expression and purification steps. The pipeline consists of a few steps in which E. coli cells are transformed with the chosen plasmid incorporating the DNA sequence of the protein of interest, selected on a plate with antibiotics, and cultured (preinoculated) in unlabeled media. Subsequently, cells are diluted in a larger volume of fresh (unlabeled or labeled) medium for further growth, and protein overexpression is induced. Maximizing the overexpression of the recombinant protein and, in addition, the selected cell district is essential to obtaining a high amount of functional recombinant protein. Once the protein has been overexpressed, it must be extracted from the cells by osmotic shock or sonication, and the solution is centrifuged to separate the insoluble part (cell membranes, heavier proteins, inclusion bodies) from the soluble part. Then, the soluble protein of interest will undergo one or more purification steps using classical chromatographic techniques (affinity, ion exchange, hydrophobic interactions, size exclusion, etc.), and its concentration will be checked by UV-Vis spectroscopy or by colorimetric assay. It should be specified that if the protein of interest is overexpressed in the insoluble part due to its toxicity and is located within the inclusion bodies (IBs), it should be extracted with a high concentration of guanidine or urea after washing and cleaning the IBs; then, the protein should be folded and purified as a classical protein. Protein folding is a critical step, as the protein must reassume its native folding at the lowest energy state. However, through gel filtration or other classical chromatographic techniques, it is typically possible to separate the different isoforms of the same protein and eventually obtain a good amount of folded protein.

O.U. P.I.: Marco Fragai

People Involved: Tommaso Staderini

Application: Protein Production


Incubator shakers
Incubation shakers are often used for cell culture, cell aeration, and solubility studies. These instruments incorporate oxygen and evenly distribute nutrients in the culture medium. In addition to providing stable temperature conditions, agitation incubators use variable-speed orbital stirring to influence cell culture growth.

ÄKTA Pure and Akta Go
ÄKTA pure machines are flexible but robust systems that allow the user and trained laboratory staff members to manage many purification needs. The system flexibility allows the user to extend the functionality of the machines with additional valves, single- or multi-wavelength detectors, fraction collectors, inlets, outlets and the use of a huge number of column for protein purification that can ensure an high rate of protein purity in short time and, furthermore, face a wide range of purification protocol. The presence of 4 pumps, divided in 2 lines (A and B) and of the mixer, allows the user to mix the buffers in order to equilibrate and to elute the sample both in isocratic and gradient (linear or step).