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BackgroundEscherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose.ResultsThe effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness.ConclusionsCWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

Le proteine prive di una struttura terziaria stabile sono definite proteine intrinsecamente disordinate (IDP). La risultante plasticità conformazionale conferisce alle IDP l’abilità di interagire con numerosi partner. Di conseguenza, le IDP sono coinvolte nelle cascate di trasduzione del segnale e nei fenomeni di condensazione proteica (detti separazione di fase liquido-liquido), i quali originano i cosiddetti “organelli privi di membrana” (MLO). Gli MLO includono svariati condensati citoplasmatici e nucleari che svolgono funzioni biologiche cruciali nella cellula. Le IDP mantengono il proprio network di interazioni principalmente attraverso contatti elettrostatici, essendo le loro catene particolarmente ricche di residui cationici ed anionici. La distribuzione delle cariche opposte (o patterning delle cariche) è emersa come caratteristica chiave responsabile delle dimensioni complessive e della propensione alla condensazione delle IDP. Un parametro di sequenza, k, è stato introdotto per descrivere in maniera quantitativa il patterning delle cariche nelle catene polipeptidiche. Investigare l’importanza del patterning delle cariche in relazione alla conformazione delle IDP e alla separazione di fase è l’obiettivo principale di questa tesi, che si organizza in due sezioni. Nella prima sezione, è stato studiato l’effetto della distribuzione delle cariche sull’ensemble conformazionale di IDP modello, determinando parallelamente l’influenza del contenuto di proline e degli elementi di struttura secondaria. Tre regioni intrinsecamente disordinate modello (IDR), ovvero NTAIL, PNT4 e NFM, similmente cariche ma con differente contenuto di proline, sono state permutate così da ottenere, a partire da ciascuna sequenza wild-type (wt), due varianti di k che presentano una differente distribuzione dei residui carichi: un costrutto “Low k”, che mostra un’alternanza più regolare rispetto alla proteina wt dei residui dotati di carica opposta; una variante “High k” con una se, Proteins lacking a stable tertiary structure are defined as intrinsically disordered proteins (IDPs). The resulting conformational plasticity gives IDPs the ability to rapidly and reversibly respond to environmental changes and to interact with multiple partners, acquiring an ordered structure upon binding or maintaining fuzzy conformations. As a consequence, IDPs are largely involved in signal transduction cascades and protein condensation phenomena (designated as liquid-liquid phase separation), which give rise to the so-called “membraneless organelles” (MLOs). MLOs include many cytoplasmatic and nuclear condensates, such as stress granules and the nucleolus, which exert crucial biological functions in the cell. IDPs maintain this interaction network primarily through electrostatic contacts, being their chains particularly enriched in cationic and anionic residues, together with prolines and glycines. Besides the net charge of the proteins, the distribution of opposite charges (or charge patterning) has emerged as a key feature dictating the overall size and condensation propensity of IDPs. A sequence parameter, k, has been introduced to quantitatively describe charge patterning in polypeptide chains. Investigating the relevance of charge patterning on IDP conformation and phase separation is the main objective of this thesis, which is organized into two sections. In the first section, the effect of charge distribution on the conformational ensemble of model IDPs was studied, in parallel assessing the influence of other sequence features such as proline content and secondary structure elements. Three model intrinsically disordered regions (IDRs), namely NTAIL, PNT4 and NFM, similarly charged yet with different proline fractions, were permutated in order to obtain from each wild-type (wt) sequence two k -variants with different distributions of charged residues: a “Low-k" variant, with a more regular alternation of oppositely charged residues than in the wt protei

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.

Broadly used in biocatalysis as acyl acceptors or (co)-solvents, short-chain alcohols often cause irreversible loss of enzyme activity. Understanding the mechanisms of inactivation is a necessary step toward the optimization of biocatalytic reactions and the design of enzyme-based sustainable processes. The functional and structural responses of an immobilized enzyme, Novozym 435 (N-435), exposed to methanol, ethanol, and tert-butanol, are explored in this work. N-435 consists of Candida antarctica lipase B (CALB) adsorbed on polymethacrylate beads and finds application in a variety of processes involving the presence of short-chain alcohols. The nature of the N-435 material required the development of an ad hoc method of structural analysis, based on Fourier transform infrared microspectroscopy, which was complemented by catalytic activity assays and by morphological observation by transmission electron microscopy. The inactivation of N-435 was found to be highly dependent on alcohol concentration and occurs through two different mechanisms. Short-chain alcohols induce conformational changes leading to CALB aggregation, which is only partially prevented by immobilization. Moreover, alcohol modifies the texture of the solid support promoting the enzyme release. Overall, knowledge of the molecular mechanisms underlying N-435 inactivation induced by short-chain alcohols promises to overcome the limitations that usually occur during industrial processes.

Protein aggregation is associated with an increasing number of human disorders and premature aging. Moreover, it is a central concern in the manufacturing of recombinant proteins for biotechnological and therapeutic applications. Nevertheless, the unique architecture of protein aggregates is also exploited by nature for functional purposes, from bacteria to humans. The relevance of this process in health and disease has boosted the interest in understanding and controlling aggregation, with the concomitant development of a myriad of algorithms aimed to predict aggregation propensities. However, most of these programs are blind to the protein environment and, in particular, to the influence of the pH. Here, we developed an empirical equation to model the pH-dependent aggregation of intrinsically disordered proteins (IDPs) based on the assumption that both the global protein charge and lipophilicity depend on the solution pH. Upon its parametrization with a model IDP, this simple phenomenological approach showed unprecedented accuracy in predicting the dependence of the aggregation of both pathogenic and functional amyloidogenic IDPs on the pH. The algorithm might be useful for diverse applications, from large-scale analysis of IDPs aggregation properties to the design of novel reversible nanofibrillar materials

The abundance of intrinsic disorder in the protein realm and its role in a variety of physiological and pathological cellular events have strengthened the interest of the scientific community in understanding the structural and dynamical properties of intrinsically disordered proteins (IDPs) and regions (IDRs). Attempts at rationalizing the general principles underlying both conformational properties and transitions of IDPs/IDRs must consider the abundance of charged residues (Asp, Glu, Lys, and Arg) that typifies these proteins, rendering them assimilable to polyampholytes or polyelectrolytes. Their conformation strongly depends on both the charge density and distribution along the sequence (i.e., charge decoration) as highlighted by recent experimental and theoretical studies that have introduced novel descriptors. Published experimental data are revisited herein in the frame of this formalism, in a new and possibly unitary perspective. The physicochemical properties most directly affected by charge density and distribution are compaction and solubility, which can be described in a relatively simplified way by tools of polymer physics. Dissecting factors controlling such properties could contribute to better understanding complex biological phenomena, such as fibrillation and phase separation. Furthermore, this knowledge is expected to have enormous practical implications for the design, synthesis, and exploitation of bio-derived materials and the control of natural biological processes.

Biocatalysis in mixtures of water and co-solvents represents an opportunity to expand the application of enzymes. However, in the presence of organic solvents, enzymes can undergo reversible inhibition, inactivation, or aggregation. In this work, we studied the effects of three co-solvents (methanol, acetone, and dimethyl sulfoxide - DMSO) on the function and structure of the recombinant Candida antarctica lipase B (rCALB), a widely used enzyme in biotechnological applications. The effects of co-solvents on rCALB were investigated by steady-state kinetics experiments, biophysical assays and by molecular dynamics simulations in the presence and upon incubation with the three co-solvents. Methanol and acetone were found to act as competitive inhibitors of rCALB and to promote its aggregation, whereas DMSO is a non-essential activator of rCALB

Psychrophilic organisms adapted to cold environments produce molecules of relevance for biotechnological application, in particular enzymes active at low temperatures and ice-binding proteins that control the growth of ice crystals. The use of cold-active enzymes supports low temperature processes that preserve heat labile compounds and can result, in some circumstances, in energy saving. Among the several possible applications in biotransformations, this paper focuses on reactions of relevance for the food industry and in molecular biology, representative of different market segments. Ice-binding proteins reduce tissues damage provoked by ice crystals and are therefore of relevance for frozen foods and for the cryopreservation of organs and tissues in the biomedical sector.