Time filter

Source Type

Santa Fe de la Vera Cruz, Argentina

Martinez-Ceron M.C.,University of Buenos Aires | Marani M.M.,University of Buenos Aires | Taules M.,University of Barcelona | Etcheverrigaray M.,Laboratorio Of Cultivos Celulares | And 3 more authors.
ACS Combinatorial Science | Year: 2011

Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. © 2011 American Chemical Society.

Mufarrege E.F.,Laboratorio Of Cultivos Celulares | Antuna S.,Laboratorio Of Cultivos Celulares | Etcheverrigaray M.,Laboratorio Of Cultivos Celulares | Kratje R.,Laboratorio Of Cultivos Celulares | Prieto C.,Laboratorio Of Cultivos Celulares
Protein Expression and Purification | Year: 2014

Background Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 5′UTR (5′ untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. Results By combining the human cytomegalovirus (CMV) or the elongation factor 1α (EF-1α) promoters along with different 5′UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 5′UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1α promoter in three widely used cell lines. Conclusion In the present work, LVs containing different promoters and 5′UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production. © 2013 Elsevier Ltd. All rights reserved.

Discover hidden collaborations