Publications by authors named "Norma A Valdez-Cruz"

33 Publications

83 increases productivity and quality of tomato ( L.): Pre and postharvest assessment.

Curr Res Microb Sci 2021 Dec 21;2:100076. Epub 2021 Oct 21.

Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca 62210, Morelos, México.

spp. are well known plant growth promoting bacteria (PGPB) and biological control agents (BCA) due to their capacity to synthesize a wide variety of phytostimulant and antimicrobial compounds. 83 is a strain marketed in Mexico as a foliar biofungicide (Fungifree AB™) which has been used for biological control of five different genera of phytopathogenic fungi () in crops of agricultural importance such as mango, avocado, papaya, citrus, tomato, strawberry, blueberry, blackberry and cucurbits, among others. In this work, the potential of plant growth promotion of 83 was evaluated on different phenological stages of tomato plants as well as the biocontrol efficacy of 83 formulations (cells and/or metabolites) against infection on leaves and postharvest fruits. Greenhouse grown tomato plants inoculated with a high concentration (1 × 10 CFU/plant) of 83 yielded 254 tons/Ha•year of which the 64% was first quality tomato (≥100 g/fruit), while the control plants produced less than 184 tons/Ha•year with only 55% of first quality tomato. Additionally, assays carried out with leaves and fruits, shown that the 83 cells formulation had an efficacy of control of infection of ∼31% on leaves and ∼89% on fruits, while the metabolites formulation had an efficacy of control of less than 10%. Therefore, it was concluded that spores (not the metabolites) are the main antagonism factor of Fungifree AB™. The high effectivity of control on fruits by 83, opens the possibility for a postharvest use of this biofungicide.
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http://dx.doi.org/10.1016/j.crmicr.2021.100076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610353PMC
December 2021

Compartmentalized Proteomic Profiling Outlines the Crucial Role of the Classical Secretory Pathway during Recombinant Protein Production in Chinese Hamster Ovary Cells.

ACS Omega 2021 May 3;6(19):12439-12458. Epub 2021 May 3.

Programa de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán 04510 Ciudad de México, México.

Different cellular processes that contribute to protein production in Chinese hamster ovary (CHO) cells have been previously investigated by proteomics. However, although the classical secretory pathway (CSP) has been well documented as a bottleneck during recombinant protein (RP) production, it has not been well represented in previous proteomic studies. Hence, the significance of this pathway for production of RP was assessed by identifying its own proteins that were associated to changes in RP production, through subcellular fractionation coupled to shot-gun proteomics. Two CHO cell lines producing a monoclonal antibody with different specific productivities were used as cellular models, from which 4952 protein groups were identified, which represent a coverage of 59% of the Chinese hamster proteome. Data are available via ProteomeXchange with identifier PXD021014. By using SAM and ROTS algorithms, 493 proteins were classified as differentially expressed, of which about 80% was proposed as novel targets and one-third were assigned to the CSP. Endoplasmic reticulum (ER) stress, unfolded protein response, calcium homeostasis, vesicle traffic, glycosylation, autophagy, proteasomal activity, protein synthesis and translocation into ER lumen, and secretion of extracellular matrix components were some of the affected processes that occurred in the secretory pathway. Processes from other cellular compartments, such as DNA replication, transcription, cytoskeleton organization, signaling, and metabolism, were also modified. This study gives new insights into the molecular traits of higher producer cells and provides novel targets for development of new sub-lines with improved phenotypes for RP production.
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http://dx.doi.org/10.1021/acsomega.0c06030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154153PMC
May 2021

Integrative overview of antibodies against SARS-CoV-2 and their possible applications in COVID-19 prophylaxis and treatment.

Microb Cell Fact 2021 Apr 22;20(1):88. Epub 2021 Apr 22.

Programa de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510, Ciudad de México, México.

SARS-CoV-2 is a novel β-coronavirus that caused the COVID-19 pandemic disease, which spread rapidly, infecting more than 134 million people, and killing almost 2.9 million thus far. Based on the urgent need for therapeutic and prophylactic strategies, the identification and characterization of antibodies has been accelerated, since they have been fundamental in treating other viral diseases. Here, we summarized in an integrative manner the present understanding of the immune response and physiopathology caused by SARS-CoV-2, including the activation of the humoral immune response in SARS-CoV-2 infection and therefore, the synthesis of antibodies. Furthermore, we also discussed about the antibodies that can be generated in COVID-19 convalescent sera and their associated clinical studies, including a detailed characterization of a variety of human antibodies and identification of antibodies from other sources, which have powerful neutralizing capacities. Accordingly, the development of effective treatments to mitigate COVID-19 is expected. Finally, we reviewed the challenges faced in producing potential therapeutic antibodies and nanobodies by cell factories at an industrial level while ensuring their quality, efficacy, and safety.
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http://dx.doi.org/10.1186/s12934-021-01576-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061467PMC
April 2021

Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights.

FEMS Microbiol Rev 2021 Nov;45(6)

Unidad de Bioprocesos, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, CP 04510, Ciudad de México, México.

Recombinant protein (RP) production from Escherichia coli has been extensively studied to find strategies for increasing product yields. The thermoinducible expression system is commonly employed at the industrial level to produce various RPs, which avoids the addition of chemical inducers, thus minimizing contamination risks. Multiple aspects of the molecular origin and biotechnological uses of its regulatory elements (pL/pR promoters and cI857 thermolabile repressor) derived from bacteriophage λ provide knowledge to improve the bioprocesses using this system. Here, we discuss the main aspects of the potential use of the λpL/pR-cI857 thermoinducible system for RP production in E. coli, focusing on the approaches of investigations that have contributed to the advancement of this expression system. Metabolic and physiological changes that occur in the host cells caused by heat stress and RP overproduction are also described. Therefore, the current scenario and the future applications of systems that use heat to induce RP production are discussed to understand the relationship between the activation of the bacterial heat shock response, RP accumulation and its possible aggregation to form inclusion bodies.
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http://dx.doi.org/10.1093/femsre/fuab023DOI Listing
November 2021

Enrichment of microsomes from Chinese hamster ovary cells by subcellular fractionation for its use in proteomic analysis.

PLoS One 2020 25;15(8):e0237930. Epub 2020 Aug 25.

Programa de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, Ciudad de México, México.

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237930PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447005PMC
October 2020

Nutrient supplementation strategy improves cell concentration and longevity, monoclonal antibody production and lactate metabolism of Chinese hamster ovary cells.

Bioengineered 2020 12;11(1):463-471

Programa de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Coyoacán, Ciudad De México, México.

A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 10 cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry.
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http://dx.doi.org/10.1080/21655979.2020.1744266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161567PMC
December 2020

From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora.

Fungal Biol 2020 Mar - Apr;124(3-4):205-218. Epub 2020 Feb 21.

Instituto de Biología, Universidad Nacional Autónoma de México, A.P. 70-233, Ciudad de México, C.P. 04510, Mexico. Electronic address:

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (Ka) of 36 h. Under those conditions less biomass (12-37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.
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http://dx.doi.org/10.1016/j.funbio.2020.02.003DOI Listing
April 2021

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

Microb Cell Fact 2019 Nov 14;18(1):200. Epub 2019 Nov 14.

Programa de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, Mexico City, CP. 04510, Mexico.

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant 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 anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents 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 concentration and pH values of contaminated media that affect 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 efficient application in treating contaminated water. Recently, stability, efficiency, 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 different sources, their structures and biochemical properties, mechanisms of action, and application 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.
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http://dx.doi.org/10.1186/s12934-019-1248-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854816PMC
November 2019

Recombinant production of ESAT-6 antigen in thermoinducible Escherichia coli: the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies.

Cell Stress Chaperones 2019 07 4;24(4):777-792. Epub 2019 Jun 4.

Programa de Investigación de Producción de Biomoléculas, Unidad de Bioprocesos, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, CP. 04510, Ciudad de México, Mexico.

The heat-inducible expression system has been widely used to produce recombinant proteins in Escherichia coli. However, the rise in temperature affects cell growth, activates the bacterial Heat-Shock Response (HSR), and promotes the formation of insoluble protein aggregates known as inclusion bodies (IBs). In this work, we evaluate the effect of the culture scale (shake flasks and bioreactors) and induction temperature (39 and 42 °C) on the kinetic behavior of thermoinducible recombinant E. coli ATCC 53606 producing rESAT-6 (6-kDa early-secretory antigenic target from Mycobacterium tuberculosis), compared with cultures grown at 30 °C (without induction). Also, the expression of the major E. coli chaperones (DnaK and GroEL) was analyzed. We found that almost twice maximum biomass and rESAT-6 production were obtained in bioreactors (~ 3.29 g/L of biomass and ~ 0.27 g/L of rESAT-6) than in shake flasks (~ 1.41 g/L of biomass and ~ 0.14 g/L of rESAT-6) when induction was carried out at 42 °C, but similar amounts of rESAT-6 were obtained from cultures induced at 39 °C (~ 0.14 g/L). In all thermo-induced conditions, rESAT-6 was trapped in IBs. Furthermore, DnaK was preferably expressed in the soluble fraction, while GroEL was present in IBs. Importantly, IBs formed at 39 °C, in both shake flasks and bioreactors, were more susceptible to degradation by proteinase-K, indicating a lower amyloid content compared to IBs formed at 42 °C. Our work presents evidence that the culture scale and the induction temperature modify the E. coli metabolic response, expression of chaperones, and structure of the IBs during rESAT-6 protein production in a thermoinducible system.
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http://dx.doi.org/10.1007/s12192-019-01006-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629757PMC
July 2019

A Novel Phospholipase A2 Isolated from Possesses Neurotoxic Activity.

Toxins (Basel) 2019 02 1;11(2). Epub 2019 Feb 1.

Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Ciudad de México C.P. 04510, Mexico.

Zoanthids of the genus are distributed worldwide in shallow waters around coral reefs. Like all cnidarians, they possess nematocysts that contain a large diversity of toxins that paralyze their prey. This work was aimed at isolating and functionally characterizing a cnidarian neurotoxic phospholipase named A2-PLTX-Pcb1a for the first time. This phospholipase was isolated from the venomous extract of the zoanthid . This enzyme, which is Ca-dependent, is a 149 amino acid residue protein. The analysis of the A2-PLTX-Pcb1a sequence showed neurotoxic domain similitude with other neurotoxic sPLA₂´s, but a different catalytic histidine domain. This is remarkable, since A2-PLTX-Pcb1a displays properties like those of other known PLA₂ enzymes.
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http://dx.doi.org/10.3390/toxins11020089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409743PMC
February 2019

Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection.

Microb Cell Fact 2019 Jan 19;18(1):11. Epub 2019 Jan 19.

Programa de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, CP. 04510, Ciudad de México, Mexico.

Background: Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein.

Results: The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb.

Conclusions: rPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.
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http://dx.doi.org/10.1186/s12934-019-1059-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339365PMC
January 2019

The metabolic switch can be activated in a recombinant strain of Streptomyces lividans by a low oxygen transfer rate in shake flasks.

Microb Cell Fact 2018 Nov 28;17(1):189. Epub 2018 Nov 28.

Programa de Investigación de Producción de Biomoléculas, Unidad de Bioprocesos, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, CP. 04510, Ciudad de México, Mexico.

Background: In Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures.

Results: Shake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF.

Conclusion: Although the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process.
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http://dx.doi.org/10.1186/s12934-018-1035-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260694PMC
November 2018

Bacterial inclusion bodies are industrially exploitable amyloids.

FEMS Microbiol Rev 2019 01;43(1):53-72

Institut de Biotecnologia i de Biomedicina (IBB), Carrer de la Vall Moronta s/n, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain.

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.
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http://dx.doi.org/10.1093/femsre/fuy038DOI Listing
January 2019

High glucose and low specific cell growth but not mild hypothermia improve specific r-protein productivity in chemostat culture of CHO cells.

PLoS One 2018 16;13(8):e0202098. Epub 2018 Aug 16.

School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile.

In the biopharmaceutical sector, Chinese hamster ovary (CHO) cells have become the host of choice to produce recombinant proteins (r-proteins) due to their capacity for correct protein folding, assembly, and posttranslational modification. However, the production of therapeutic r-proteins in CHO cells is expensive and presents insufficient production yields for certain proteins. Effective culture strategies to increase productivity (qp) include a high glucose concentration in the medium and mild hypothermia (28-34 °C), but these changes lead to a reduced specific growth rate. To study the individual and combined impacts of glucose concentration, specific growth rate and mild hypothermia on culture performance and cell metabolism, we analyzed chemostat cultures of recombinant human tissue plasminogen activator (rh-tPA)-producing CHO cell lines fed with three glucose concentrations in feeding media (20, 30 and 40 mM), at two dilution rates (0.01 and 0.018 1/h) and two temperatures (33 and 37 °C). The results indicated significant changes in cell growth, cell cycle distribution, metabolism, and rh-tPA productivity in response to the varying environmental culture conditions. High glucose feed led to constrained cell growth, increased specific rh-tPA productivity and a higher number of cells in the G2/M phase. Low specific growth rate and temperature (33 °C) reduced glucose consumption and lactate production rates. Our findings indicated that a reduced specific growth rate coupled with high feed glucose significantly improves r-protein productivity in CHO cells. We also observed that low temperature significantly reduced qp, but not cell growth when dilution rate was manipulated, regardless of the glucose concentration or dilution rate. In contrast, we determined that feed glucose concentration and consumption rate were the dominant aspects of the growth and productivity in CHO cells by using multivariate analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202098PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095543PMC
January 2019

Recombinant-phospholipase A2 production and architecture of inclusion bodies are affected by pH in Escherichia coli.

Int J Biol Macromol 2018 Mar 31;108:826-836. Epub 2017 Oct 31.

Programa de Investigación de Producción de Biomoléculas, Unidad de Bioprocesos, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Ciudad de México, México. Electronic address:

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15-25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.10.178DOI Listing
March 2018

Production of a recombinant phospholipase A2 in Escherichia coli using resonant acoustic mixing that improves oxygen transfer in shake flasks.

Microb Cell Fact 2017 Jul 25;16(1):129. Epub 2017 Jul 25.

Programa de Investigación de Producción de Biomoléculas, Unidad de Bioprocesos, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, CP 04510, Mexico City, Mexico.

Background: Shake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom.

Results: Comparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (ka ≈ 80 h) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response.

Conclusions: In moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.
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http://dx.doi.org/10.1186/s12934-017-0746-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526256PMC
July 2017

Effect of Temperature Downshift on the Transcriptomic Responses of Chinese Hamster Ovary Cells Using Recombinant Human Tissue Plasminogen Activator Production Culture.

PLoS One 2016 18;11(3):e0151529. Epub 2016 Mar 18.

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de México, México.

Recombinant proteins are widely used as biopharmaceuticals, but their production by mammalian cell culture is expensive. Hence, improvement of bioprocess productivity is greatly needed. A temperature downshift (TDS) from 37°C to 28-34°C is an effective strategy to expand the productive life period of cells and increase their productivity (qp). Here, TDS in Chinese hamster ovary (CHO) cell cultures, initially grown at 37°C and switched to 30°C during the exponential growth phase, resulted in a 1.6-fold increase in the qp of recombinant human tissue plasminogen activator (rh-tPA). The transcriptomic response using next-generation sequencing (NGS) was assessed to characterize the cellular behavior associated with TDS. A total of 416 (q > 0.8) and 3,472 (q > 0.9) differentially expressed transcripts, with more than a 1.6-fold change at 24 and 48 h post TDS, respectively, were observed in cultures with TDS compared to those at constant 37°C. In agreement with the extended cell survival resulting from TDS, transcripts related to cell growth arrest that controlled cell proliferation without the activation of the DNA damage response, were differentially expressed. Most upregulated genes were related to energy metabolism in mitochondria, mitochondrial biogenesis, central metabolism, and avoidance of apoptotic cell death. The gene coding for rh-tPA was not differentially expressed, but fluctuations were detected in the transcripts encoding proteins involved in the secretory machinery, particularly in glycosylation. Through NGS the dynamic processes caused by TDS were assessed in this biological system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151529PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798216PMC
August 2016

Positive effect of reduced aeration rate on growth and stereospecificity of DL-malic acid consumption by Azospirillum brasilense: improving the shelf life of a liquid inoculant formulation.

J Biotechnol 2015 Feb 30;195:74-81. Epub 2014 Dec 30.

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP 70228, México, D.F. CP 04510, Mexico. Electronic address:

Azospirillum brasilense has significance as a growth promoter in plants of commercial interest. Two industrial native strains (Start and Calf), used as a part of an inoculant formulation in Mexico during the last 15 years, were incubated in laboratory-scale pneumatic bioreactors at different aeration rates. In both strains, the positive effect of decreased aeration was observed. At the lowest (0.1 vvm, air volume/liquid volume×minute), the highest biomass were obtained for Calf (7.8 × 10(10)CFU/ml), and Start (2.9 × 10(9)CFU/ml). These were higher in one magnitude order compared to cultures carried out at 0.5 vvm, and two compared to those at 1.0 vvm. At lower aeration, both stereoisomeric forms of malic acid were consumed, but at higher aeration, just L-malate was consumed. A reduction in aeration allows an increase of the shelf life and the microorganism saved higher concentrations of polyhydroxybutyrate. The selected fermentation conditions are closely related to those prevalent in large-scale bioreactors and offer the possibility of achieving high biomass titles with high shelf life at a reduced costs, due to the complete use of a carbon source at low aeration of a low cost raw material as DL-malic acid mixture in comparison with the L-malic acid stereoisomer.
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http://dx.doi.org/10.1016/j.jbiotec.2014.12.020DOI Listing
February 2015

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli.

Microb Cell Fact 2014 Sep 12;13:137. Epub 2014 Sep 12.

Background: Inclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies.

Results: Uncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and β-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented β-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding.

Conclusions: The manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.
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http://dx.doi.org/10.1186/s12934-014-0137-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177172PMC
September 2014

Conservation of the mycelia of the medicinal mushroom Humphreya coffeata (Berk.) Stey. in sterile distilled water.

MethodsX 2014 9;1:19-22. Epub 2014 May 9.

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP 70228, CP 04510, México, D.F., México.

Currently, there is a growing interest in obtaining and studying the biologically active compounds from higher basidiomycetes, such as Ganoderma lucidum, Lentinus edodes and Inonotus obliquus[1], but the techniques for safe long-term storage are time-consuming, susceptible to contamination, and do not prevent genetic and physiological changes during long-term maintenance [2]. A recent strategy for obtaining biologically active compounds is using mycelia submerged cultures of these mushrooms, cultured under controlled laboratory conditions [1]. However, obtaining spores of these fungi under these conditions is difficult, and in most cases the way to obtain the spores is unknown [1]. Therefore, the strategy for mycelium storage seems to be more appropriated and simple.•A modification of Castellani's method [3-7] is proposed for higher basidiomycetes, by using the mycelium of Humphreya coffeata (Berk.) Stey., whose culture filtrates demonstrated bioactivity against lymphoma cells [8].•H. coffeata (Berk.) Stey. was grown on malt extract agar with filter paper disks that were removed after 4 days, placed in tubes with sterile distilled water, and stored at 4 °C.•Filter paper disks with H. coffeata (Berk.) Stey. stored at 4 °C were confirmed to be viable for up to 18 months, with no visible morphological alterations.
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http://dx.doi.org/10.1016/j.mex.2014.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472852PMC
July 2015

Scale-up from shake flasks to pilot-scale production of the plant growth-promoting bacterium Azospirillum brasilense for preparing a liquid inoculant formulation.

Appl Microbiol Biotechnol 2013 Nov 6;97(22):9665-74. Epub 2013 Sep 6.

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, Mexico, D.F., 04510, Mexico,

Azospirillum brasilense has industrial significance as a growth promoter in plants of commercial interest. However, there is no report in the literature disclosing a liquid product produced in pilot-scale bioreactors and is able to be stored at room temperature for more than 2 years. The aim of this work was to scale up a process from a shake flask to a 10-L lab-scale and 1,000-L pilot-scale bioreactor for the production of plant growth-promoting bacterium A. brasilense for a liquid inoculant formulation. Furthermore, this work aimed to determine the shelf life of the liquid formulation stored at room temperature and to increase maize crops yield in greenhouses. Under a constant oxygen mass transfer coefficient (K L a), a fermentation process was successfully scaled up from shake flasks to 10- and 1,000-L bioreactors. A concentration ranging from 3.5 to 7.5 × 10(8) CFU/mL was obtained in shake flasks and bioreactors, and after 2 years stored at room temperature, the liquid formulation showed one order of magnitude decrease. Applications of the cultured bacteria in maize yields resulted in increases of up to 95 % in corncobs and 70 % in aboveground biomass.
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http://dx.doi.org/10.1007/s00253-013-5199-9DOI Listing
November 2013

Scale-up from shake flasks to bioreactor, based on power input and Streptomyces lividans morphology, for the production of recombinant APA (45/47 kDa protein) from Mycobacterium tuberculosis.

World J Microbiol Biotechnol 2013 Aug 10;29(8):1421-9. Epub 2013 Mar 10.

Unidad de Bioprocesos, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, Mexico, DF, CP. 04510, Mexico.

Culture conditions in shake flasks affect filamentous Streptomyces lividans morphology, as well the productivity and O-mannosylation of recombinant Ala-Pro-rich O-glycoprotein (known as the 45/47 kDa or APA antigen) from Mycobacterium tuberculosis. In order to scale up from previous reported shake flasks to bioreactor, data from the literature on the effect of agitation on morphology of Streptomyces strains were used to obtain gassed volumetric power input values that can be used to obtain a morphology of S. lividans in bioreactor similar to the morphology previously reported in coiled/baffled shake flasks by our group. Morphology of S. lividans was successfully scaled-up, obtaining similar mycelial sizes in both scales with diameters of 0.21 ± 0.09 mm in baffled and coiled shake flasks, and 0.15 ± 0.01 mm in the bioreactor. Moreover, the specific growth rate was successfully scaled up (0.09 ± 0.02 and 0.12 ± 0.01 h(-1), for bioreactors and flasks, respectively), and the recombinant protein productivity measured by densitometry, as well. More interestingly, the quality of the recombinant glycoprotein measured as the amount of mannoses attached to the C-terminal of APA was also scaled- up; with up to five mannose residues in cultures carried out in shake flasks; and six in the bioreactor. However, final biomass concentration was not similar, indicating that although the process can be scaled-up using the power input, others factors like oxygen transfer rate, tip speed or energy dissipation/circulation function can be an influence on bacterial metabolism.
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http://dx.doi.org/10.1007/s11274-013-1305-5DOI Listing
August 2013

Isolation, amino acid sequence and biological characterization of an "aspartic-49" phospholipase A₂ from Bothrops (Rhinocerophis) ammodytoides venom.

Toxicon 2012 Dec 10;60(7):1314-23. Epub 2012 Sep 10.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Apartado Postal 510-3, Cuernavaca Morelos 62210, Mexico.

A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD₅₀ of this phospholipase in CD-1 mice was around 6 μg/g of mouse body weight (more exactly 117 μg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 μg/g. In contrast, the LD₅₀ of the venom was circa 2 μg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.
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http://dx.doi.org/10.1016/j.toxicon.2012.08.019DOI Listing
December 2012

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.

Microb Cell Fact 2011 Dec 20;10:110. Epub 2011 Dec 20.

Unidad de Bioprocesos, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP, 70228, México, D,F,, CP, 04510, México.

Background: The Ala-Pro-rich O-glycoprotein known as the 45/47 kDa or APA antigen from Mycobacterium tuberculosis 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 O-glycoprotein APA was produced by the non-pathogenic filamentous bacteria Streptomyces lividans, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to M. tuberculosis.

Results: Three different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of S. lividans 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 O-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.

Conclusions: The shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the O-mannosylation of rAPA in S. lividans. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant O-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.
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http://dx.doi.org/10.1186/1475-2859-10-110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266650PMC
December 2011

Molecular responses of Escherichia coli caused by heat stress and recombinant protein production during temperature induction.

Bioeng Bugs 2011 Mar-Apr;2(2):105-10

Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, A.P. 70228, México, D.F., C.P. 04510, México.

In a recent review, we discussed the extensively used temperature-inducible expression system, based on the pL and/or pR phage lambda promoters that are finely regulated by the thermo-labile cI857 repressor. In this system, an increase in temperature induces the heterologous protein production and activates the heat shock response, as well as the stringent and SOS responses. The same responses are activated just by the overproduction of recombinant protein. All such responses result in a metabolic burden to the cells, a decrease in the specific growth rate, and alterations in the central carbon metabolism. Altogether, these effects can alter the quantity and quality of the produced foreign protein. Here, we compare and discuss the transcription of selected genes, and the concomitant synthesis of heat-shock proteins (hsp) soon after thermal induction, in relation to the responses that occur in other expression systems that also trigger the heat-shock response.
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http://dx.doi.org/10.4161/bbug.2.2.14316DOI Listing
September 2011

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

Microb Cell Fact 2010 Mar 19;9:18. Epub 2010 Mar 19.

Unidad de Bioprocesos, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México, DF, México.

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 prokaryotic cells. In this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37 degrees 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 lambda 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.
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http://dx.doi.org/10.1186/1475-2859-9-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848208PMC
March 2010

[Hydrodynamic stress: death and cellular damage in agitated cultures].

Rev Latinoam Microbiol 2006 Jul-Dec;48(3-4):269-80

PROBIOMED SA de CV Departamento de Investigación y Desarrollo, Col.Granada, México DF, México.

All submerged cultures are under forces associated to fluid dinamics. In this review, the bases of hydrodynamic stress in agitated systems of submerged cultures will be presented, starting from the definitions of eddy of microscale, shear stress and shear rate. This work reports as well some of the most important proposals of cellular response of cells affected by shear injuries, and how other stress response mechanisms allow the cell to respond to the hydrodynamic effects. In addition, some of the most remarkable reports in each one of the cellular culture models will be presented that can be affected by the mechanical agitation, such as: bacteria, filamentous fungi, vegetal and animal cells.
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April 2008

Sequence analysis and phylogenetic relationship of genes encoding heterodimeric phospholipases A2 from the venom of the scorpion Anuroctonus phaiodactylus.

Gene 2007 Jul 24;396(1):149-58. Epub 2007 Mar 24.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Apartado Postal 510-3, Cuernavaca 622106221, Mexico.

Some scorpion venom contain heterodimeric phospholipases A2. They were shown to be toxic to insects and to cause edema and/or hemolysis of mammalian erythrocytes. This manuscript describes the results of cDNA cloning of five different heterodimeric phospholipases from the venomous glands of the Mexican scorpion Anuroctonus phaiodactylus. The amino acid sequence deduced from the heterodimeric phospholipases open reading frames corresponds in each case to a different isoform. The nucleotide sequences corresponding to two of these genes were also obtained by directly sequencing genomic DNA. The cDNA isoforms show high similarity with the heterodimeric phospholipase Phaiodactylipin purified from the same scorpion. However, similar phospholipases were also found in scorpions from other species and the sequences available were used to construct a phylogenetic tree. In order to understand better the gene structure and phylogeny of these enzymes we analyzed their sequences and compared them with secretory phospholipases of other sources from groups I, II and III. The genomic DNA sequence of a similar phospholipase from bee venomous glands was also cloned. The information available on a Drosophila phospholipase was included in this analysis. The phospholipases of groups I and II contain a conserved exon-intron structure (four or five exons of the mature segment of the enzyme are separated by three or four introns). Also, the gene structure of the phospholipases from A. phaiodactylus and that of the bee venom, belonging to group III phospholipases, are interrupted by three introns. The mature peptide of the bee enzyme is a single polypeptide chain, coded by four exons, whereas those from the scorpion studied here although having four exons, showed the presence of two different polypeptides in its native state. The mature protein is processed after synthesis, producing the heterodimeric structure: a long and a short-peptide chain, linked by a disulfide bridge. The small subunit is the one coded by the fourth exon. The human phospholipase A2 and that of Drosophila, also classified into the group III phospholipases, have a considerably different exon-intron organization.
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http://dx.doi.org/10.1016/j.gene.2007.03.007DOI Listing
July 2007

Anuroctoxin, a new scorpion toxin of the alpha-KTx 6 subfamily, is highly selective for Kv1.3 over IKCa1 ion channels of human T lymphocytes.

Mol Pharmacol 2005 Apr 22;67(4):1034-44. Epub 2004 Dec 22.

Department of Biophysics and Cell Biology, University of Debrecen, Medical and Health Science Center, 98 Nagyerdei krt., Debrecen, Hungary 4012.

The physiological function of T lymphocytes can be modulated selectively by peptide toxins acting on Kv1.3 K(+) channels. Because Kv1.3-specific peptide toxins are considered to have a significant therapeutic potential in the treatment of autoimmune diseases, the discovery of new toxins is highly motivated. Through chromatographic procedures and electrophysiological assays, using patch-clamp methodology, the isolation of a novel peptide named anuroctoxin was accomplished using the venom of the Mexican scorpion Anuroctonus phaiodactylus. It has 35 amino acid residues with a molecular weight of 4082.8, tightly bound by four disulfide bridges whose complete covalent structure was determined. It has a pyroglutamic acid at the N-terminal region and an amidated C-terminal residue. Sequence comparison and phylogenetic clustering analysis classifies anuroctoxin into subfamily 6 of the alpha-KTx scorpion toxins (systematic name, alpha-KTx 6.12). Patch-clamp experiments show that anuroctoxin is a high-affinity blocker of Kv1.3 channels of human T lymphocytes with a K(d) of 0.73 nM, and it does not block the Ca(2+)-activated IKCa1 K(+) channels. These two channels play different but important roles in T-lymphocyte activation. Furthermore, the toxin practically does not inhibit Shaker IR, mKv1.1, and rKv2.1 channels, whereas the affinity of anuroctoxin for hKv1.2 is almost an order of magnitude smaller than for Kv1.3. The pharmacological profile and the selectivity of this new toxin for Kv1.3 over IKCa1 may provide an important tool for the modulation of the immune system, especially in cases in which selective inhibition of Kv1.3 is required.
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http://dx.doi.org/10.1124/mol.104.007187DOI Listing
April 2005

Phaiodotoxin, a novel structural class of insect-toxin isolated from the venom of the Mexican scorpion Anuroctonus phaiodactylus.

Eur J Biochem 2004 Dec;271(23-24):4753-61

Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Cuernavaca, Mexico.

A peptide called phaiodotoxin was isolated from the venom of the scorpion Anuroctonus phaiodactylus. It is lethal to crickets, but non toxic to mice at the doses assayed. It has 72 amino acid residues, with a molecular mass of 7971 atomic mass units. Its covalent structure was determined by Edman degradation and mass spectrometry; it contains four disulfide-bridges, of which one of the pairs is formed between cysteine-7 and cysteine-8 (positions Cys63-Cys71). The other three pairs are formed between Cys13-Cys38, Cys23-Cys50 and Cys27-Cys52. Comparative sequence analysis shows that phaiodotoxin belongs to the long-chain subfamily of scorpion peptides. Several genes coding for this peptide and similar ones were cloned by PCR, using cDNA prepared from the RNA of venomous glands of this scorpion. Electrophysiological assays conducted with this toxin in several mammalian cell lines (TE671, COS7, rat GH3 and cerebellum granular cells), showed no effect on Na+ currents. However, it shifts the voltage dependence of activation and inactivation of insect Na+ channels (para/tipE) to more negative and positive potentials, respectively. Therefore, the 'window' current is increased by 225%, which is thought to be the cause of its toxicity toward insects. Phaiodotoxin is the first toxic peptide ever purified from a scorpion of the family Iuridae.
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http://dx.doi.org/10.1111/j.1432-1033.2004.04439.xDOI Listing
December 2004
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