Chemical modifications in the seed region of miRNAs 221/222 increase the silencing performances in gastrointestinal stromal tumor cells.

Most GastroIntestinal Stromal Tumors (GISTs) are characterized by KIT gene overexpression, which in turn is regulated by levels of microRNA 221 and microRNA 222. GISTs can also be distinguished by their miRNAs expression profile in which miRNAs 221/222 result reduced in comparison with GI normal tissues. In this paper, to restore normal miRNAs levels and to improve the silencing performances of miRNAs 221/222, new miRNA mimics in which guide strands are modified by Phosphorothioate (PS) and/or 2'-O-methyl RNA (2'-OMe) inside and outside the seed region, were synthesized and tested in GIST48 cells.

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen®.

Background: Filgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.

Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency.

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency.

A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization.

We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH(2)-terminal sequence and peptide mapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim.

Enzymatic mono-pegylation of glucagon-like peptide 1 towards long lasting treatment of type 2 diabetes.

Human glucagon-like peptide-1 (GLP-1) is a physiological gastrointestinal peptide with glucose-dependent insulinotropic effects which is therefore considered an interesting antidiabetic agent. However, after in vivo administration, exogenous GLP-1 does not exert its physiological action due to the combination of rapid proteolytic degradation by ubiquitous dipeptidyldipeptidase IV (DPP IV) enzyme and renal clearance resulting in an extremely short circulating half-life. In this work we describe the conjugation of GLP-1-(7-36)-amide derivatives with polyethylene glycol (PEG) by enzymatic site-specific transglutamination reaction as an approach to reduce both the proteolysis and the renal clearance rates.

Efficient bacterial expression of fusion proteins and their selective processing by a recombinant Kex-1 protease.

A secreted, soluble variant of the Kex-1 endopeptidase from Kluyveromyces lactis has been produced and studied as a novel cleavage enzyme exhibiting high specificity for the Lys-Arg peptide. This highly selective, efficient enzyme is particularly adapted for use in manufacturing when a recombinant therapeutic protein, possessing its native N-terminus, has to be released in vitro from a bacterially-expressed fusion protein. In this paper, we describe the preparation of a Kex-1 variant using Saccharomyces cerevisiae and its application in the production of important therapeutic recombinant proteins such as human growth hormone, granulocyte colony-stimulating factor and interferon-alpha-2b.

Characterization of Escherichia coli uridine phosphorylase by single-site mutagenesis.

The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results.

FT-IR study of heterologous protein expression in recombinant Escherichia coli strains.

Two recombinant Escherichia coli strains expressing different levels of an interferon fusion protein as inclusion bodies have been studied by Fourier transform infrared (FT-IR) microspectroscopy. A marker band at 1628 cm(-1) allowed monitoring of the protein expression by direct analysis of cell pellets in a rapid, non-invasive and quantitative way. The results demonstrate that FT-IR microspectroscopy is a technique of potential biotechnological interest for studying inclusion body formation.