Mixing and mass transfer in shake flasks and their relationship with antibodies productivity of CHO cells

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Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters

The temperature inducible expression system, based on the pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor has been widely use to produce recombinant proteins in prokariotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37°C. Concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. Many studies have reported the use of such temperature inducible expression system, however only few discuss the simultaneous stress effects caused by recombinant protein production and the up-shift in temperature. Understanding the integral effect of such responses should be useful to develop improved strategies for high yield protein production and recovery. Here, we describe the current status of the heat inducible expression system based on the pL and/or pR λ phage promoters, focusing on recent developments on expression vehicles, the stress responses at the molecular and physiological level that occur after heat induction, and bioprocessing factors that affect protein overexpression, including culture operation variables and induction strategies

Subcellular fractionation coupled to shotgun proteomics allows the identification of novel targets of the classical secretion pathway associated with increased productivity in recombinant CHO cells

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The O-mannosylation and production of recombinant APA (45/47 KDa) protein from Mycobacterium tuberculosis in Streptomyces lividans is affected by culture conditions in shake flasks

<p>Abstract</p> <p>Background</p> <p>The Ala-Pro-rich <it>O</it>-glycoprotein known as the 45/47 kDa or APA antigen from <it>Mycobacterium tuberculosis </it>is an immunodominant adhesin restricted to mycobacterium genus and has been proposed as an alternative candidate to generate a new vaccine against tuberculosis or for diagnosis kits. In this work, the recombinant <it>O</it>-glycoprotein APA was produced by the non-pathogenic filamentous bacteria <it>Streptomyces lividans</it>, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to <it>M. tuberculosis</it>.</p> <p>Results</p> <p>Three different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of <it>S. lividans </it>and the production of rAPA was characterized and evaluated. Small unbranched free filaments and mycelial clumps were found in baffled and coiled shake flasks, but one order of magnitude larger pellets were found in conventional shake flasks. The production of rAPA is around 3 times higher in small mycelia than in larger pellets, most probably due to difficulties in mass transfer inside pellets. Moreover, there are four putative sites of <it>O</it>-mannosylation in native APA, one of which is located at the carboxy-terminal region. The carbohydrate composition of this site was determined for rAPA by mass spectrometry analysis, and was found to contain different glycoforms depending on culture conditions. Up to two mannoses residues were found in cultures carried out in conventional shake flasks, and up to five mannoses residues were determined in coiled and baffled shake flasks.</p> <p>Conclusions</p> <p>The shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the <it>O</it>-mannosylation of rAPA in <it>S. lividans</it>. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant <it>O</it>-glycosylated proteins with similar "quality" in bacteria, particularly, if the protein activity depends on the glycosylation pattern. Furthermore, it will be an interesting exercise to determine the effect of shear and oxygen in shake flasks, to obtain evidences that may be useful in scaling-up these processes to bioreactors. Another approach will be using lab-scale bioreactors under well-controlled conditions, and study the impact of those on rAPA productivity and quality.</p

Teaching bioreactors operation and scale-up of bioprocesses: Multidisciplinary training in a theoretical-practical course of one week

La biotecnología es un área emergente y prioritaria para países en vías de desarrollo. Los biorreactores son un equipo fundamental y se convierten en un elemento clave para el desarrollo e innovación en bioprocesos. El conocimiento de estos equipos es fundamental para lograr el crecimiento y desarrollo de microorganismos, células animales y vegetales, e inclusive la micropropagación de plantas. En una iniciativa conjunta por parte de los autores de este artículo y con el apoyo de las respectivas instituciones académicas, desde 2012 el Curso Internacional “Escalado de Bioprocesos y Entrenamiento en Operación de Biorreactores” se ha realizado por siete ocasiones. El objetivo del curso es ofrecer a la comunidad científica de Iberoamérica un entrenamiento Teórico - Práctico en bioprocesos. Producto de este curso, se ha capacitado a más de 1400 personas a través de conferencias y a 216 personas con las habilidades prácticas en el laboratorio. El curso ha permitido fomentar una red de contactos que operan en la consultoría y en la movilidad de personas. Además, uno de los productos destacados es la integración de un libro, que contribuye como una obra de consulta en el tema.Biotechnology is an emerging and priority area for developing countries. Bioreactors are essential equipment and a key element for development and innovation in bioprocesses. The knowledge of this equipment is essential to achieve the growth of microorganisms, animal and plant cells, and even the micropropagation of plants. In a joint initiative by the authors of this article, and with the support of the respective academic institutions, the International Course “Bioprocess Scaling and Training in Bioreactor Operation” has been held seven times since 2012. The objective of this course is to offer to the Latin American scientific community a theoretical and practical training in bioprocesses. As a result of this course, more than 1400 people have been trained through lectures and 216 people with practical skills in the laboratory. The course has promoted a network of contacts that operate in consulting and people mobility. In addition to the fact that one of the outstanding products is the integration of a book.Fil: Trujillo Roldán, Mauricio A.. Universidad Nacional Autónoma de México; MéxicoFil: Orozco Sánchez, Fernando. Universidad Nacional de Colombia. Sede Medellín; ColombiaFil: Valdez Cruz, Norma A.. Universidad Nacional Autónoma de México; MéxicoFil: Rodríguez Monroy, Mario. Instituto Politécnico Nacional. Centro de Desarrollo de Productos Bióticos; MéxicoFil: Busto, Víctor Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; Argentin

Bacterial inclusion bodies are industrially exploitable amyloids

Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.info:eu-repo/semantics/acceptedVersio

Computational Design of Inhibitors Targeting the Catalytic β Subunit of Escherichia coli FOF1-ATP Synthase

With the uncontrolled growth of multidrug-resistant bacteria, there is an urgent need to search for new therapeutic targets, to develop drugs with novel modes of bactericidal action. FoF1-ATP synthase plays a crucial role in bacterial bioenergetic processes, and it has emerged as an attractive antimicrobial target, validated by the pharmaceutical approval of an inhibitor to treat multidrug-resistant tuberculosis. In this work, we aimed to design, through two types of in silico strategies, new allosteric inhibitors of the ATP synthase, by targeting the catalytic β subunit, a centerpiece in communication between rotor subunits and catalytic sites, to drive the rotary mechanism. As a model system, we used the F1 sector of Escherichia coli, a bacterium included in the priority list of multidrug-resistant pathogens. Drug-like molecules and an IF1-derived peptide, designed through molecular dynamics simulations and sequence mining approaches, respectively, exhibited in vitro micromolar inhibitor potency against F1. An analysis of bacterial and Mammalia sequences of the key structural helix-turn-turn motif of the C-terminal domain of the β subunit revealed highly and moderately conserved positions that could be exploited for the development of new species-specific allosteric inhibitors. To our knowledge, these inhibitors are the first binders computationally designed against the catalytic subunit of FOF1-ATP synthase. Keywords: FOF1-ATP synthase; allosteric inhibition; evolutionary and PPI algorithms; peptide design; structure-based drug design

Bacterial inclusion bodies are industrially exploitable amyloids

Altres ajuts: CERCA Programme/Generalitat de CatalunyaUnderstanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology

Laccases: structure, function, and potential application in water bioremediation

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign con‑ taminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropo‑ genic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifcally, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the efuents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentra‑ tion and pH values of contaminated media that afect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efcient application in treating contaminated water. Recently, stability, efciency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from diferent sources, their structures and biochemical properties, mechanisms of action, and applica‑ tion in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.Instituto de Fisiología Vegeta