Membrane-anchored calpains - hidden regulators of growth and development beyond plants?

Calpains are modulatory proteases that modify diverse cellular substrates and play essential roles in eukaryots. The best studied are animal cytosolic calpains. Here, we focus on enigmatic membrane-anchored calpains, their structural and functional features as well as phylogenetic distribution. Based on domain composition, we identified four types of membrane-anchored calpains. Type 1 and 2 show broad phylogenetic distribution among unicellular protists and streptophytes suggesting their ancient evolutionary origin. Type 3 and 4 diversified early and are present in brown algae and oomycetes. The plant DEK1 protein is the only representative of membrane-anchored calpains that has been functionally studied. Here, we present up to date knowledge about its structural features, putative regulation, posttranslational modifications, and biological role. Finally, we discuss potential model organisms and available tools for functional studies of membrane-anchored calpains with yet unknown biological role. Mechanistic understanding of membrane-anchored calpains may provide important insights into fundamental principles of cell polarization, cell fate control, and morphogenesis beyond plants.

Impact of media components from different suppliers on enterokinase productivity in Pichia pastoris.

BACKGROUND: The aim of this study was to provide an information about the homogeneity on the level of enterokinase productivity in P. pastoris depending on different suppliers of the media components. RESULTS: In previous studies, we performed the optimisation process for the production of enterokinase by improving the fermentation process. Enterokinase is the ideal enzyme for removing fusion partners from target recombinant proteins. In this study, we focused our optimization efforts on the sources of cultivation media components. YPD media components were chosen as variables for these experiments. Several suppliers for particular components were combined and the optimisation procedure was performed in 24-well plates. Peptone had the highest impact on enterokinase production, where the difference between the best and worst results was threefold. The least effect on the production level was recorded for yeast extract with a 1.5 fold difference. The worst combination of media components had a activity of only 0.15 U/ml and the best combination had the activity of 0.88 U/ml, i.e., a 5.87 fold difference. A substantially higher impact on the production level of enterokinase was observed during fermentation in two selected media combinations, where the difference was almost 21-fold. CONCLUSIONS: Results demonstrated in the present study show that the media components from different suppliers have high impact on enterokinase productivity and also provide the hypothesis that the optimization process should be multidimensional and for achieving best results it is important to perform massive process also in terms of the particular media component supplier .

The ploidy determination of the biotechnologically important yeast Candida utilis.

Yeast Candida utilis is considered to be a potentially advantageous expression system for production of recombinant proteins utilizable for industrial and pharmaceutical purposes. As the scientific literature is not consistent in the ploidy of this yeast, in this work, we focused on resolving the problem via several methods such as the copy number determination of maltase gene by multiplex PCR, measuring α-glucosidase activity, the characterization of maltase gene copy number in deletion mutants using qPCR and flow cytometry. In context with the published data and results obtained in this study about the copy number of the maltase gene on C. utilis genome, we attempted to hypothesise and made conclusion about the ploidy of C. utilis. The results of this work, besides the biotechnological aspect, contribute to the elementary knowledge of C. utilis. The exact information about the ploidy or more specifically about the copy number of appropriate gene is essential for expression cassette dosage determination integrated into the chromosome of the host. In this study, we come to the conclusion that the maltase gene is present in C. utilis genome in four alleles, and in combination with flow cytometry, published information and the published genome sequences, the observations support the theory about tetraploidy of C. utilis.

Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris.

Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains.

Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations.