Use of disposable reactors to generate inoculum cultures for E. coli production fermentations

Disposable technology is being used more each year in the biotechnology industry. Disposable bioreactors allow one to avoid expenses associated with cleaning, assembly and operations, as well as equipment validation. The WAVE bioreactor is well established for Chinese Hamster Ovary (CHO) production, however, it has not yet been thoroughly tested for E. coli production because of the high oxygen demand and temperature maintenance requirements of that platform. The objective of this study is to establish a robust process to generate inoculum for E. coli production fermentations in a WAVE bioreactor. We opted not to evaluate the WAVE system for production cultures because of the high cell densities required in our current E. coli production processes. Instead, the WAVE bioreactor 20/50 system was evaluated at laboratory scale (10‐L) to generate inoculum with target optical densities (OD₅₅₀) of 15 within 7–9 h (pre‐established target for stainless steel fermentors). The maximum settings for rock rate (40 rpm) and angle (10.5) were used to maximize mass transfer. The gas feed was also supplemented with additional oxygen to meet the high respiratory demand of the culture. The results showed that the growth profiles for the inoculum cultures were similar to those obtained from conventional stainless steel fermentors. These inoculum cultures were subsequently inoculated into 10‐L working volume stainless steel fermentors to evaluate the inocula performance of two different production systems during recombinant protein production. The results of these production cultures using WAVE inocula showed that the growth and recombinant protein production was comparable to the control data set. Furthermore, an economic analysis showed that the WAVE system would require less capital investment for installation and operating expenses would be less than traditional stainless steel systems. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.     

A short survey on protein blocks

Protein structures are classically described in terms of secondary structures. Even if the regular secondary structures have relevant physical meaning, their recognition from atomic coordinates has some important limitations such as uncertainties in the assignment of boundaries of helical and β-strand regions. Further, on an average about 50% of all residues are assigned to an irregular state, i.e., the coil. Thus different research teams have focused on abstracting conformation of protein backbone in the localized short stretches. Using different geometric measures, local stretches in protein structures are clustered in a chosen number of states. A prototype representative of the local structures in each cluster is generally defined. These libraries of local structures prototypes are named as "structural alphabets". We have developed a structural alphabet, named Protein Blocks, not only to approximate the protein structure, but also to predict them from sequence. Since its development, we and other teams have explored numerous new research fields using this structural alphabet. We review here some of the most interesting applications.     

Protein determination by ponceau S using digital color image analysis of protein spots on nitrocellulose membranes

A procedure was developed to estimate protein concentrations using color image analysis of protein spots stained with ponceau S. The method involved spotting a constant volume (2 microl) of the protein solutions on nitrocellulose paper, staining with acidic ponceau S, destaining, and air drying the paper. The image of the nitrocellulose paper was grabbed using a digital color scanner and thresholded with an optimal value to mark the area of the spot. The intensity of the color in the spot was measured in an arbitrary unit of intensity termed as inverse integrated gray value. This value showed a discernible increase with protein concentrations from 0.1 to 50 microg protein per spot (0.05-25 mg/ml). The method is simple and convenient compared to the conventional spectrophotometric procedures and allows several samples to be analyzed simultaneously. It can also be used to estimate protein concentration in the spots stained with Coomassie brilliant blue or other dyes.     

Comparative role of proteins in transport of HCH-isomers in desert locust, Schistocerca gregaria Forskal and bovines

The studies on binding of hexachlorocyclohexane (HCH) with carrier proteins were carried out to establish the role of proteins in the transport of insecticides in insects. Sephadex G-200 column chromatography resolved haemolymph of adult male desert locust, Schistocerca gregaria into three major protein peaks. There was significant binding of gamma-HCH with first protein peak (F1). Two classes of binding sites were observed on first protein peak for gamma-HCH. However low level of binding was observed with the third protein peak (F3) of the haemolymph. Bindings of HCH-isomers (alpha, beta and gamma) with bovine serum albumin (BSA) were not related to their water solubilities. Moderate to low affinities (1.4 -1.84 x 10(6) M(-1)) of HCH-isomers for BSA were observed. The present studies showed that more HCH binds to haemolymph lipoprotein of locust as compared to BSA. This indicates a significant role of haemolymph proteins in the transport of insecticides in insects.     

Fast response temperature measurement and highly reproducible heating methods for 96-well plates

Hyperthermia, the procedure of exposing cells to a temperature between 42 degrees and 49 degrees C, has been shown to be a promising approach for cancer treatment. To understand the underlying mechanisms of hyperthermic killing of cancer cells, it is critical to have an accurate temperature measurement technique and a heating method with high reproducibility. To this end, we have developed a method using fine thermocouples with fast response time to measure the temperatures in multiple wells of a 96-well plate. The accuracy of temperature measurement was +/- 0.2 degree C. Such a capability allows a complete record of the time and temperature of the treatment procedure and helps define an accurate thermal dose. We have also compared several methods for heating 96-well plates and found that use of copper blocks in contact with the lower surface of the 96-well plate in an incubator provides a highly reproducible heating method. The common method of using water bath to heat cells in vitro resulted in a decrease of cell viability even at the control temperature of 37 degrees C and a decrease in the reproducibility of certain biological assays. In summary, using these improved techniques, proposed thermal dose can be defined more precisely, and highly reproducible heating in vitro can be achieved.     

An SH2 domain-dependent,phosphotyrosine-independent interaction between Vav1 and the Mer receptor tyrosine kinase: a mechanism for localizing guanine nucleotide-exchange factor action

Mer belongs to the Mer/Axl/Tyro3 receptor tyrosine kinase family, which regulates immune homeostasis in part by triggering monocyte ingestion of apoptotic cells. Mutations in Mer can also cause retinitis pigmentosa, again due to defective phagocytosis of apoptotic material. Although, some functional aspects of Mer have been deciphered, how receptor activation lead to the physiological consequences is not understood. By using yeast two-hybrid assays, we identified the carboxyl-terminal region of the guanine nucleotide-exchange factor (GEF) Vav1 as a Mer-binding partner. Unlike similar (related) receptors, Mer interacted with Vav1 constitutively and independently of phosphotyrosine, yet the site of binding localized to the Vav1 SH2 domain. Mer activation resulted in tyrosine phosphorylation of Vav1 and release from Mer, whereas Vav1 was neither phosphorylated nor released from kinase-dead Mer. Mutation of the Vav1 SH2 domain phosphotyrosine coordinating Arg-696 did not alter Mer/Vav1 constitutive binding or Vav1 tyrosine phosphorylation but did retard Vav1 release from autophosphorylated Mer. Ligand-dependent activation of Mer in human monocytes led to Vav1 release and stimulated GDP replacement by GTP on RhoA family members. This unusual constitutive, SH2 domain-dependent, but phosphotyrosine-independent, interaction and its regulated local release and subsequent activation of Rac1, Cdc42, and RhoA may explain how Mer coordinates precise cytoskeletal changes governing the ingestion of apoptotic material by macrophages and pigmented retinal epithelial cells.     

Mer receptor tyrosine kinase signaling: prevention of apoptosis and alteration of cytoskeletal architecture without stimulation or proliferation

Mer is a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family, a family whose physiological function is not well defined. We constructed a Mer chimera using the epidermal growth factor receptor (EGFR) extracellular and transmembrane domains and the Mer cytoplasmic domain. Stable transfection of the Mer chimera into interleukin 3 (IL-3)-dependent murine 32D cells resulted in ligand-activable surface receptor that tyrosine autophosphorylated, stimulated intracellular signaling, and dramatically reduced apoptosis initiated by IL-3 withdrawal. However, unlike multiple other ectopically expressed receptor tyrosine kinases including full-length EGFR or an EGFR/Axl chimera, the Mer chimera did not stimulate proliferation. Moreover, and in contrast to EGFR, Mer chimera activation induced adherence and cell flattening in the normally suspension-growing 32D cells. The Mer chimera signal also blocked IL-3-dependent proliferation leading to G(1)/S arrest, dephosphorylation of retinoblastoma protein, and elongation of cellular processes. Unlike other agonists that lead to a slow (4-8 days) ligand-dependent differentiation of 32D cells, the combined Mer and IL-3 signal resulted in differentiated morphology and growth cessation in the first 24 h. Thus the Mer chimera blocks apoptosis without stimulating growth and produces cytoskeletal alterations; this outcome is clearly separable from the proliferative signal produced by most receptor tyrosine kinases.     

Assessment of copper and zinc status in hair and urine of young women descendants of NIDDM parents

The concentration of copper (Cu) and zinc (Zn) in hair and urine were studied in young nonpregnant healthy women whose both parents were diagnosed for noninsulin-dependent diabetes mellitus (NIDDM descendants) and were compared with those of young healthy nonpregnant females with no family history of NIDDM or hypertension (non-NIDDM descendants) and NIDDM patients. The concentration of Zn in hair in NIDDM descendants was significantly higher than that of non-NIDDM descendants (p < 0.001) and insignificantly higher than that of NIDDM patients. The hair Cu concentrations in NIDDM descendant and patients were significantly lower than that of non-NIDDM descendants (p < 0.001). Hyperzincuria was detected in some NIDDM patients and hypocuperuria in all NIDDM descendants and patients. The data suggest that the young healthy NIDDM descendants possess high-Zn and low-Cu reserves in their bodies, and the observed perturbation appears to be associated with Cu-Zn antagonism.