Norma A. Valdez-Cruz

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.

A large number of novel Ergtoxin-like genes and ERG K+-channels blocking peptides from scorpions of the genus Centruroides

Twenty‐three novel sequences similar to Ergtoxin (ErgTx) were obtained by direct sequencing of peptides or deduced from gene cloned using cDNAs of venomous glands of Centruroides (C.) elegans, C. exilicauda, C. gracilis, C. limpidus limpidus, C. noxius and C. sculpturatus. These peptides have from 42 to 47 amino acid residues cross‐linked by four disulfide bridges. They share sequence similarities (60–98% compared with ErgTx1) and were shown to block ERG K+‐channels of F‐11 clone (N18TG‐2×rat DRG) cultured cells. An unrooted phylogenetic tree analysis of these peptides showed that they conform at least five different subfamilies, of which three are novel subfamilies.

Genes and peptides from the scorpion Centruroides sculpturatus Ewing, that recognize Na(+)-channels.

Sixteen different genes were cloned from the venomous glands of Centruroides sculpturatus Ewing using RNA extracted from scorpions collected in Tucson, Arizona. Based on the amino acid sequence similarities of the proteins coded by these genes, all together there are 22 different structural components in this venom, thought to be specific for Na(+)-channels. The genes reported contain signal peptides with 19 amino acid residues followed by mature peptides of 63-66 amino acid residues in length. One of them correspond to toxin I (CsEI), a known scorpion toxin specific for Na(+)-channels. Four different genes are almost identical to variant 1 (Csv1), presenting only one amino acid change from the original protein. For variant 2 (Csv2) four related genes were found, with only one amino acid change in their primary sequences. Another gene resembles to variant 3 (Csv3, the best known Centruroides sculpturatus toxin), with only three amino acid changes in their primary sequences. Additionally, two genes show variations only on the nucleotide sequence at level of the signal peptides, and several genes clearly show sequences that suggest post-transcriptional modifications, during the maturation process. A phylogenetic tree was generated with the primary structures available and three main divergent branches were found.

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

BackgroundInclusion 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.ResultsUncontrolled 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.ConclusionsThe 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.

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

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.

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

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.

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

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.

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

Graphical abstract

Co-Expression of the Mosquitocidal Toxins Cyt1Aa and Cry11Aa from Bacillus thuringiensis Subsp. israelensis in Asticcacaulis excentricus

The cyt1Aa gene from Bacillus thuringiensis subsp. israelensis (Bti), whose product synergizes other mosquitocidal toxins, and functions as a repressor of resistance developed by mosquitoes against Bacilli insecticides, was introduced into the aquatic Gram-negative bacterium Asticcacaulis excentricus alongside the cry11Aa gene. The genes were introduced as an operon, but although mRNA was detected for both genes, no Cyt1Aa toxin was detected. Both proteins were expressed using a construct in which a promoter was inserted upstream of each gene. Recombinant A. excentricus expressing both toxins was found to be approximately twice as toxic to third instar larvae of Culex quinquefasciatus as transformants expressing just Cry11Aa.

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

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.