Agustín Correa

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease

Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Tuning different expression parameters to achieve soluble recombinant proteins in E. coli: Advantages of high‐throughput screening

Proteins are the main reagents for structural, biomedical, and biotechnological studies; however, some important challenges remain concerning protein solubility and stability. Numerous strategies have been developed, with some success, to mitigate these challenges, but a universal strategy is still elusive. Currently, researchers face a plethora of alternatives for the expression of the target protein, which generates a great diversity of conditions to be evaluated. Among these, different promoter strength, diverse expression host and constructs, or special culture conditions have an important role in protein solubility. With the arrival of automated high‐throughput screening (HTS) systems, the evaluation of hundreds of different conditions within reasonable cost and time limits is possible. This technology increases the chances to obtain the target protein in a pure, soluble, and stable state. This review focuses on some of the most commonly used strategies for the expression of recombinant proteins in the enterobacterium Escherichia coli, including the use of HTS for the production of soluble proteins.

Phylogenetic analysis of bovine leukemia viruses isolated in South America reveals diversification in seven distinct genotypes

Bovine leukaemia virus (BLV) is an oncogenic member of the genus Deltaretrovirus of the family Retroviridae. Recent studies revealed that BLV strains can be classified into six different genotypes and raised the possibility that another genotype may exist. In order to gain insight into the degree of genetic variability of BLV strains circulating in the South American region, a phylogenetic analysis was performed using gp51 env gene sequences. The results of these studies revealed the presence of seven BLV genotypes in this geographic region and the suitability of partial gp51 env gene sequences for phylogenetic inference. A significant number of amino acid substitutions found in BLV strains isolated in South America map to the second neutralization domain of gp51. A 3D molecular model of BLV gp51 revealed that these substitutions are located on the surface of the molecule. This may provide a selective advantage to overcome immune host neutralization.

Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Overcoming the Solubility Problem in E. coli : Available Approaches for Recombinant Protein Production

Despite the importance of recombinant protein production in academy and industrial fields, many issues concerning the expression of soluble and homogeneous product are still unsolved. Although several strategies were developed to overcome these obstacles, at present there is no magic bullet that can be applied for all cases. Indeed, several key expression parameters need to be evaluated for each protein. Among the different hosts for protein expression, Escherichia coli is by far the most widely used. In this chapter, we review many of the different tools employed to circumvent protein insolubility problems.

Structure of a human IgA1 Fab fragment at 1.55 Å resolution: potential effect of the constant domains on antigen-affinity modulation.

Despite being the most abundant class of immunoglobulins in humans and playing central roles in the adaptive immune response, high-resolution structural data are still lacking for the antigen-binding region of human isotype A antibodies (IgAs). The crystal structures of a human Fab fragment of IgA1 in three different crystal forms are now reported. The three-dimensional organization is similar to those of other Fab classes, but FabA1 seems to be more rigid, being constrained by a hydrophobic core in the interface between the variable and constant domains of the heavy chain (VH-CH1) as well as by a disulfide bridge that connects the light and heavy chains, influencing the relative heavy/light-chain orientation. The crystal structure of the same antibody but with a G-isotype CH1 which is reported to display different antigen affinity has also been solved. The differential structural features reveal plausible mechanisms for constant/variable-domain long-distance effects whereby antibody class switching could alter antigen affinity.

Generation of a vector suite for protein solubility screening

Recombinant protein expression has become an invaluable tool for academic and biotechnological projects. With the use of high-throughput screening technologies for soluble protein production, uncountable target proteins have been produced in a soluble and homogeneous state enabling the realization of further studies. Evaluation of hundreds conditions requires the use of high-throughput cloning and screening methods. Here we describe a new versatile vector suite dedicated to the expression improvement of recombinant proteins (RP) with solubility problems. This vector suite allows the parallel cloning of the same PCR product into the 12 different expression vectors evaluating protein expression under different promoter strength, different fusion tags as well as different solubility enhancer proteins. Additionally, we propose the use of a new fusion protein which appears to be a useful solubility enhancer. Above all we propose in this work an economic and useful vector suite to fast track the solubility of different RP. We also propose a new solubility enhancer protein that can be included in the evaluation of the expression of RP that are insoluble in classical expression conditions.

In Search of Topical Agricultural Biofungicides: Properties of the Recombinant Antimicrobial Peptide TrxAq-AMP Obtained from Amaranthus quitensis

Synthetic pesticides have a positive impact on food production. However, there are concerns due to the outbreak of resistance along with negative side effects on human health and the environment. New active compounds and control strategies are needed for the management of phytopathogens. Antimicrobial peptides (AMPs) are evolutionarily conserved components of the innate immune response in almost all organisms that constitute an interesting source of potential molecules for use as pesticides. The naturally derived antimicrobial peptide Aq-AMP (Amaranthus quitensis-Antimicrobial Peptide), obtained from Amaranthus quitensis, is cysteine-rich with activity against several phytopathogens. In the present work, we report on the expression in Escherichia coli of functionally active Aq-AMP fused to thioredoxin (TrxAq-AMP). The in vitro antifungal activity of purified TrxAq-AMP was confirmed against Alternaria solani, Fusarium oxysporum f. sp. lycopersici, Pencillium digitatum and P. italicum, as well as the in vivo control of P. digitatum in oranges. We demonstrated the stability of TrxAq-AMP in a range of pH (from 3 to 11) and at temperatures from 0°C to 100°C. Furthermore, it maintained activity after digestion with various proteases and it displayed no haemolytic activity suggesting a highly stable and safe molecule. For topical application we present an AMP with gathering no hemolytic/phytotoxic activity that is effective, stable to a wide range of temperature and pH, pH-conditions and resistant to protease activity. Besides, this molecule is naturally stored in the seed, easily to extract and potentially produced through molecular farming. These findings encourage further biotechnological research on topical application of AMPs, especially those in relation related to molecule bioavailability.

The EAL-domain protein FcsR regulates flagella, chemotaxis and type III secretion system in Pseudomonas aeruginosa by a phosphodiesterase independent mechanism

The second messenger c-di-GMP regulates the switch between motile and sessile bacterial lifestyles. A general feature of c-di-GMP metabolism is the presence of a surprisingly large number of genes coding for diguanylate cyclases and phosphodiesterases, the enzymes responsible for its synthesis and degradation respectively. However, the physiological relevance of this apparent redundancy is not clear, emphasizing the need for investigating the functions of each of these enzymes. Here we focused on the phosphodiesterase PA2133 from Pseudomonas aeruginosa, an important opportunistic pathogen. We phenotypically characterized P. aeruginosa strain K overexpressing PA2133 or its inactive mutant. We showed that biofilm formation and motility are severely impaired by overexpression of PA2133. Our quantitative proteomic approach applied to the membrane and exoprotein fractions revealed that proteins involved in three processes were mostly affected: flagellar motility, type III secretion system and chemotaxis. While inhibition of biofilm formation can be ascribed to the phosphodiesterase activity of PA2133, down-regulation of flagellar, chemotaxis, and type III secretion system proteins is independent of this enzymatic activity. Based on these unexpected effects of PA2133, we propose to rename this gene product FcsR, for Flagellar, chemotaxis and type III secretion system Regulator.

Multi-compartment and multi-host vector suite for recombinant protein expression and purification.

Recombinant protein expression has become an invaluable tool in basic and applied research. The accumulated knowledge in this field allowed the expression of thousands of protein targets in a soluble, pure, and homogeneous state, essential for biochemical and structural analyses. A lot of progress has been achieved in the last decades, where challenging proteins were expressed in a soluble manner after evaluating different parameters such as host, strain, and fusion partner or promoter strength, among others. In this regard, we have previously developed a vector suite that allows the evaluation of different promoters and solubility enhancer-proteins, through an easy and efficient cloning strategy. Nonetheless, the proper expression of many targets remains elusive, requiring, for example, the addition of complex post-translation modifications and/or passage through specialized compartments. In order to overcome the limitations found when working with a single subcellular localization and a single host type, we herein expanded our previously developed vector suite to include the evaluation of recombinant protein expression in different cell compartments and cell hosts. In addition, these vectors also allow the assessment of alternative purification strategies for the improvement of target protein yields.