De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome in cells.

Cytochrome (cyt ) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt was previously reported, but the hybridoma was rendered nonviable.

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

Despite the importance of recombinant protein production in the academy and industrial fields, many issues concerning the expression of soluble and homogeneous products are still unsolved. Several strategies were developed to overcome these obstacles; however, 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.

Production, purification and characterization of a double-tagged TEV protease.

Many recombinant proteins are products of great value in biomedical and industrial fields. The use of solubility and affinity tags are commonly used to increase yields and facilitate the purification process. However, it is of paramount importance in several applications to remove the fusion tag from the final product.

Overview of High-Throughput Cloning Methods for the Post-genomic Era.

The advent of new DNA sequencing technologies leads to a dramatic increase in the number of available genome sequences and therefore of target genes with potential for functional analysis. The insertion of these sequences into proper expression vectors requires a simple an efficient cloning method. In addition, when expressing a target protein, quite often it is necessary to evaluate different DNA constructs to achieve a soluble and homogeneous expression of the target with satisfactory yields.

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.

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.

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.

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.

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.

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.

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.

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.

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.