Rosa María Ferraz

Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins

BackgroundMany enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein.ResultsBy using two structurally and functionally different model enzymes and two fluorescent proteins we show that physiological aggregation in bacteria might only result in a moderate loss of biological activity and that inclusion bodies can be used in reaction mixtures for efficient catalysis.ConclusionThis observation offers promising possibilities for the exploration of inclusion bodies as catalysts for industrial purposes, without any previous protein-refolding step.

Insertional protein engineering for analytical molecular sensing

The quantitative detection of low analyte concentrations in complex samples is becoming an urgent need in biomedical, food and environmental fields. Biosensors, being hybrid devices composed by a biological receptor and a signal transducer, represent valuable alternatives to non biological analytical instruments because of the high specificity of the biomolecular recognition. The vast range of existing protein ligands enable those macromolecules to be used as efficient receptors to cover a diversity of applications. In addition, appropriate protein engineering approaches enable further improvement of the receptor functioning such as enhancing affinity or specificity in the ligand binding. Recently, several protein-only sensors are being developed, in which either both the receptor and signal transducer are parts of the same protein, or that use the whole cell where the protein is produced as transducer. In both cases, as no further chemical coupling is required, the production process is very convenient. However, protein platforms, being rather rigid, restrict the proper signal transduction that necessarily occurs through ligand-induced conformational changes. In this context, insertional protein engineering offers the possibility to develop new devices, efficiently responding to ligand interaction by dramatic conformational changes, in which the specificity and magnitude of the sensing response can be adjusted up to a convenient level for specific analyte species. In this report we will discuss the major engineering approaches taken for the designing of such instruments as well as the relevant examples of resulting protein-only biosensors.

Fast electrochemical detection of anti-HIV antibodies: Coupling allosteric enzymes and disk microelectrode arrays

Here a novel electrochemical method for the rapid detection of anti-HIV antibodies in serum is presented. The novelty lies in the combination of allosteric enzymes and coulometry to yield a fast, simple and reliable HIV diagnostic method. We have used a previously developed beta-galactosidase enzyme that is efficiently activated by anti-HIV antibodies directed against a major B-cell epitope of the gp41 glycoprotein. When these antibodies bind the enzyme, the 3D conformation changes positively affecting the performance of the active site and, consequently, the enzyme activity is stimulated. Using 4-aminophenyl beta-D-galactopyranoside (PAPG) as substrate yields p-aminophenol (PAP), which is reversibly oxidised at a very mild potential, ca. 0.37 V vs. Ag/AgCl over a range of electrode materials within the working pH range of beta-galactosidase. In the present case, photolithographically produced microelectrode arrays resulted in a detection limit of 4 microM for 4-aminophenol (PAP). The presence of anti-HIV antibodies results in enzyme activity increases above 50% which, combined with the sensitivity and response time afforded by the microelectrode arrays, allowed for the diagnosis of HIV in sera samples within an hour.

Inclusion bodies of fuculose-1-phosphate aldolase as stable and reusable biocatalysts.

Fuculose‐1‐phosphate aldolase (FucA) has been produced in Escherichia coli as active inclusion bodies (IBs) in batch cultures. The activity of insoluble FucA has been modulated by a proper selection of producing strain, culture media, and process conditions. In some cases, when an optimized defined medium was used, FucA IBs were more active (in terms of specific activity) than the soluble protein version obtained in the same process with a conventional defined medium, supporting the concept that solubility and conformational quality are independent protein parameters. FucA IBs have been tested as biocatalysts, either directly or immobilized into Lentikat® beads, in an aldolic reaction between DHAP and (S)‐Cbz‐alaninal, obtaining product yields ranging from 65 to 76%. The production of an active aldolase as IBs, the possibility of tailoring IBs properties by both genetic and process approaches, and the reusability of IBs by further entrapment in appropriate matrices fully support the principle of using self‐assembled enzymatic clusters as tunable mechanically stable and functional biocatalysts.

Antiretroviral Therapy-Induced Functional Modification of IgG4 and IgM Responses in HIV-1–Infected Individuals Screened by an Allosteric Biosensor

We have explored the effect of antiretroviral drugs on the antiviral immune response in human immunodeficiency virus-1 (HIV-1)—infected patients by using an enzymatic immunosensor that detects epitope-modifying anti-gp41 antibodies. By this molecular sensing approach, we have identified an irreversible impact of drug administration on the functionality of IgG4 and IgM specific antibodies regarding the structural modification promoted on their target epitope. During the antiretroviral therapy, the prevalent induced fit promoted by IgM on the epitope was lost at the expense of that promoted by IgG4, suggesting alternative-ness in the neutralization potency of these antibody subpopulations. Because the particular drug composition of the antiretroviral treatment did not affect such immune shift, the obtained data strongly suggest that the drop in the viral load and the consequent lost of antigenemia are responsible for the functional adaptation observed in the humoral response. (Journal of Biomolecular Screening 2008:817-821)

Screening HIV-1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors.

We have analyzed the suitability of six antigenic peptides from several HIV‐1 structural proteins (namely gp41, gp120, p17, and p24), as anti‐HIV‐1 antibody receptors in an allosteric enzymatic biosensor. These peptides were inserted in a solvent‐exposed surface of Escherichia coli (E. coli) beta‐galactosidase by means of conventional recombinant DNA technology. The resulting enzymes were tested to allosterically respond to sera from HIV‐1‐infected individuals. Only stretches from gp41 and gp120 envelope proteins were able to transduce the molecular contact signal in the presence of immunoreactive sera. Intriguingly, the enzyme displaying the CD4 binding site segment KQFINMWQEVGKAMYAPP was activated by soluble CD4, suggesting that it produces conformational modifications on the allosteric enzyme as those occurring during antibody‐promoted induced fit. This fact is discussed in the context of the design of smart protein drugs and markers targeted to CD4+ cells. Copyright

Efficient, antibody-mediated allosteric activation of an immobilized, E. coli beta-galactosidase recombinant biosensor

who generously supported the meeting. Meeting abstracts - A single PDF containing all abstracts in this supplement is available he re . http://www. biomedcentral.co m/content/pdf/14 75-2859-5-S1-inf o.pdf