Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products.

Biopharmaceutical products are of great importance in the treatment or prevention of many diseases and represent a growing share of the global pharmaceutical market. The usual technology for protein synthesis (cell-based expression) faces certain obstacles, especially with 'difficult-to-express' proteins. Cell-free protein synthesis (CFPS) can overcome the main bottlenecks of cell-based expression.

On the expression of recombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3) Rosetta strains.

The CRISPR-Cas9 system is a new tool that has been extensively used for genome editing. The system is composed of a Cas9 endonuclease, which has the function of cleaving DNA at a specific site, and a guide RNA (gRNA), which contains the sequence of the cleavage site that is the target of editing. Despite the great interest that has been generated because of the utility of Cas 9 as a molecular tool and a potential therapeutic protein, the production of the 158 kDa recombinant Cas9 protein derived from Streptococcus pyogenes remains a challenge.

Switching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides.

Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown.

Techno-economic analysis of the industrial production of a low-cost enzyme using : the case of recombinant β-glucosidase.

Background: The enzymatic conversion of lignocellulosic biomass into fermentable sugars is a promising approach for producing renewable fuels and chemicals. However, the cost and efficiency of the fungal enzyme cocktails that are normally employed in these processes remain a significant bottleneck. A potential route to increase hydrolysis yields and thereby reduce the hydrolysis costs would be to supplement the fungal enzymes with their lacking enzymatic activities, such as β-glucosidase.

Protein nanoparticles are nontoxic, tuneable cell stressors.

Aim: Nanoparticle-cell interactions can promote cell toxicity and stimulate particular behavioral patterns, but cell responses to protein nanomaterials have been poorly studied.

Results: By repositioning oligomerization domains in a simple, modular self-assembling protein platform, we have generated closely related but distinguishable homomeric nanoparticles. Composed by building blocks with modular domains arranged in different order, they share amino acid composition.

Intracellular trafficking of a dynein-based nanoparticle designed for gene delivery.

The success of viruses in the delivery of the viral genome to target cells relies on the evolutionary selection of protein-based domains able to hijack the intermolecular interactions through which cells respond to intra- and extracellular stimuli. In an effort to mimic viral infection capabilities during non-viral gene delivery, a modular recombinant protein named T-Rp3 was recently developed, containing a DNA binding domain, a dynein molecular motor interacting domain, and a TAT-derived transduction domain. Here, we analyzed at the microscopic level the mechanisms behind the cell internalization and intracellular trafficking of this highly efficient modular protein vector.

Microfluidic devices for continuous production of pDNA/cationic liposome complexes for gene delivery and vaccine therapy.

To evaluate the process parameters for the production of plasmid DNA/cationic liposome (pDNA/CL) complexes in microfluidic systems, we studied two microfluidic devices: one with simple straight hydrodynamic flow focusing (SMD) and a second one with barriers in the mixing microchannel (patterned walls, PMD). A conventional bulk mixing method was used as a comparison to microfluidic mixing. The CL and the pDNA were combined at a molar positive/negative charge ratio of 6.

Sodium citrate and potassium phosphate as alternative adsorption buffers in hydrophobic and aromatic thiophilic chromatographic purification of plasmid DNA from neutralized lysate.

The number of studies on gene therapy using plasmid vectors (pDNA) has increased in recent years. As a result, the demand for preparations of pDNA in compliance with recommendations of regulatory agencies (EMEA, FDA) has also increased. Plasmid DNA is often obtained through fermentation of transformed Escherichia coli and purification by a series of unit operations, including chromatography.

Cloning, expression, and purification of the virulence-associated protein D from Xylella fastidiosa.

In this study, an efficient expression system, based on the pET32Xa/LIC vector, for producing a Xylella fastidiosa virulence-associated protein D, found to have a strong similarity to Riemerella anatipestifer and Actinobacillus actinomycetencomitans VapD protein, is presented. The protein has a molecular mass of 17.637 Da and a calculated pI of 5.

Expression and purification of a putative H-NS nucleoid-associated protein from the phytopathogen Xylella fastidiosa.

The H-NS protein is one of the major constituents of the nucleoid structure that has been implicated in the DNA packaging and in the global regulation of gene expression. The study of this transcriptional regulator is an effort to fight Xylella fastidiosa, a citrus pathogen responsible for a range of economically important plant diseases, including the citrus variegated chlorosis (CVC). The putative H-NS ORF was cloned into a pET32-Xa/LIC vector in order to overexpress it coupled with fusion tags in Escherichia coli BL21(DE3).