A novel genetic system for recombinant protein secretion in the Antarctic Pseudoalteromonas haloplanktis TAC125

BACKGROUND: The final aim of recombinant protein production is both to have a high specific production rate and a high product quality. It was already shown that using cold-adapted bacteria as host vectors, some "intractable" proteins can be efficiently produced at temperature as low as 4°C. RESULTS: A novel genetic system for the production and secretion of recombinant proteins in the Antarctic Gram-negative bacterium Pseudoalteromonas haloplanktis TAC125 was set up. This system aims at combining the low temperature recombinant product production with the advantages of extra-cellular protein targeting. The psychrophilic α-amylase from Pseudoalteromonas haloplanktis TAB23 was used as secretion carrier. Three chimerical proteins were produced by fusing intra-cellular proteins to C-terminus of the psychrophilic α-amylase and their secretion was analysed. Data reported in this paper demonstrate that all tested chimeras were translocated with a secretion yield always higher than 80%. CONCLUSION: Data presented here demonstrate that the "cold" gene-expression system is efficient since the secretion yield of tested chimeras is always above 80%. These secretion performances place the α-amylase derived secretion system amongst the best heterologous secretion systems in Gram-negative bacteria reported so far. As for the quality of the secreted passenger proteins, data presented suggest that the system also allows the correct disulphide bond formation of chimera components, secreting a fully active passenger

Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

Background: In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in Pseudoalteromonas haloplanktis TAC125. This system makes use of the psychrophilic α-amylase from P. haloplanktis TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, Pseudoalteromonales are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a P. haloplanktis TAC125 mutant strain with reduced extra-cellular proteolytic activity. Results: P. haloplanktis TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of P. haloplanktis TAC125 in which the gspE gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-β-lactamase chimera. Conclusion: Here we report a cell engineering approach to the construction of a P. haloplanktis TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic α-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the gspE mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that P. haloplanktis TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium

Experimenting With Discursive and Non-Discursive Styles of Teaching Absolute Value Inequalities to Mature Students

This research is a follow-up of Sierpinska, Bobos, and Pruncut’s 2011 study, which experimented with three teaching approaches to teaching absolute value inequalities (AVI), visual, procedural, and theoretical, presented over an audio lecture with slides. The study demonstrated that participants treated with the visual approach were more likely to engage in theoretical thinking than those treated with the other two approaches. In the present experiment, two groups of participants enrolled in prerequisite mathematics courses at a large, urban North American University were taught AVI with the visual approach using two different teaching styles: discursive (permitting and actively encouraging teacher-student interactions during the lecture) and non-discursive (not allowing teacher-student interactions during the lecture). In Sierpinska et al.’s study, the non-discursive style was used in all three approaches (the lectures were recorded and the teacher was not present in person). In the present study, a live teacher was lecturing in both treatments. Another difference was that in Sierpinska et al.’s study, lectures were delivered individually to each participant, while in the present study, all participants in a group were treated simultaneously. Therefore, in the discursive approach, not only teacher-student but also student-student interactions during the lecture were possible. The aim of this research was to explore the conjecture that the discursive approach is more likely to promote theoretical thinking in students. The group exposed to the discursive approach was, therefore, my experimental group and the other played the role of the control group. The conjecture was not confirmed, but the two approaches seem to have provoked different aspects of theoretical thinking. The experimental group was found to be more reflective, while the control group tended to be more systemic in their thinking. Some striking results, not predicted by Sierpinska et al.’s study, were also found with respect to reflective thinking, definitional thinking, proving behavior, and analytic thinking

Influence of production process design on inclusion bodies protein: the case of an Antarctic flavohemoglobin

<p>Abstract</p> <p>Background</p> <p>Protein over-production in <it>Escherichia coli </it>often results in formation of inclusion bodies (IBs). Some recent reports have shown that the aggregation into IBs does not necessarily mean that the target protein is inactivated and that IBs may contain a high proportion of correctly folded protein. This proportion is variable depending on the protein itself, the genetic background of the producing cells and the expression temperature. In this paper we have evaluated the influence of other production process parameters on the quality of an inclusion bodies protein.</p> <p>Results</p> <p>The present paper describes the recombinant production in <it>Escherichia coli </it>of the flavohemoglobin from the Antarctic bacterium <it>Pseudoalteromonas haloplanktis </it>TAC125. Flavohemoglobins are multidomain proteins requiring FAD and heme cofactors. The production was carried out in several different experimental setups differing in bioreactor geometry, oxygen supply and the presence of a nitrosating compound. In all production processes, the recombinant protein accumulates in IBs, from which it was solubilized in non-denaturing conditions. Comparing structural properties of the solubilized flavohemoglobins, i.e. deriving from the different process designs, our data demonstrated that the protein preparations differ significantly in the presence of cofactors (heme and FAD) and as far as their secondary and tertiary structure content is concerned.</p> <p>Conclusions</p> <p>Data reported in this paper demonstrate that other production process parameters, besides growth temperature, can influence the structure of a recombinant product that accumulates in IBs. To the best of our knowledge, this is the first reported example in which the structural properties of a protein solubilized from inclusion bodies have been correlated to the production process design.</p

Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature

<p>Abstract</p> <p>Background</p> <p>In a previous paper, we reported the accomplishment of a cold gene-expression system for the recombinant secretion of heterologous proteins in <it>Pseudoalteromonas haloplanktis </it>TAC125. This system makes use of the psychrophilic α-amylase from <it>P. haloplanktis </it>TAB23 as secretion carrier, and allows an effective extra-cellular addressing of recombinant proteins. However, <it>Pseudoalteromonales </it>are reported to secrete a wide range of extra-cellular proteases. This feature works against the efficiency of the cold-adapted secretion system, because of the proteolytic degradation of recombinant products. The aim of this study is the construction of a <it>P. haloplanktis </it>TAC125 mutant strain with reduced extra-cellular proteolytic activity.</p> <p>Results</p> <p><it>P. haloplanktis </it>TAC125 culture medium resulted to contain multiple and heterogeneous proteases. Since the annotation of the Antarctic bacterium genome highlighted the presence of only one canonical secretion machinery, namely the Type II secretion pathway (T2SS), we have inactivated this secretion system by a gene insertion strategy. A mutant strain of <it>P. haloplanktis </it>TAC125 in which the <it>gspE </it>gene was knocked-out, actually displayed a remarkable reduction of the extra-cellular protease secretion. Quite interestingly this strain still retained the ability to secrete the psychrophilic amylase as efficiently as the wild type. Moreover, the decrease in extra-cellular proteolytic activity resulted in a substantial improvement in the stability of the secreted amylase-β-lactamase chimera.</p> <p>Conclusion</p> <p>Here we report a cell engineering approach to the construction of a <it>P. haloplanktis </it>TAC125 strain with reduced extra-cellular protease activity. The improved strain is able to secrete the psychrophilic α-amylase (the carrier of our recombinant secretion system), while it displays a significant reduction of protease content in the culture medium. These features make the <it>gspE </it>mutant an improved host with a remarkable biotechnological potential in recombinant protein secretion at low temperature. Moreover this work demonstrates that <it>P. haloplanktis </it>TAC125 is a versatile psychrophilic host for recombinant protein production since it can be easily improved by a directed engineering approach. To the best of our knowledge, this is the first described example of a strain improvement strategy applied to an Antarctic bacterium.</p

Acetate: friend or foe? Efficient production of a sweet protein in Escherichia coli BL21 using acetate as a carbon source

Escherichia coli is, to date, the most used microorganism for the production of recombinant proteins and biotechnologically relevant metabolites. High density cell cultures allow efficient biomass and protein yields. However, their main limitation is the accumulation of acetate as a by-product of unbalanced carbon metabolism. Increased concentrations of acetate can inhibit cellular growth and recombinant protein production, and many efforts have been made to overcome this problem. On the other hand, it is known that E. coli is able to grow on acetate as the sole carbon source, although this mechanism has never been employed for the production of recombinant proteins

Inhibitory effect of vitamin K1 on growth and polyamine biosynthesis of human gastric and colon carcinoma cell lines

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An improved method to determine PM-bound nitro-PAHs in ambient air

Nowadays, no a standard method for the determination of particulate bound nitro-PAHs (NPAHs) has been developed. Existing methods include complex sampling and extraction procedures. Moreover, their sensitivity does not allow to analyze daily PM10 samples, affecting the temporal resolution of NPAH concentrations. In this study an analytical method for the quantification of NPAHs on half 47 mm-filter samples of daily PM10 was developed and validated. NPAHs were recovered by microwave-assisted extraction, and analyzed by using a gas chromatography-triple quadrupole mass spectrometry in MRM mode. The analytical performance for 14 NPAHs (2-nitrofluorene, 9-nitroanthracene, 9-nitrophenantrene, 3-nitrophenantrene, 2-nitroanthracene, 3-nitrofluoranthene, 1-nitropyrene, 2,7-dinitrofluorene, 7-nitrobenzo[a]anthracene, 6-nitrochrysene, 1,3-dinitropyrene, 1,8-dinitropyrene, 1,6-dinitropyrene, 6-nitrobenza[a]pyrene) was investigated. Recovery extraction percentage exceeded 95% for all target compounds in the range between 0.25 and 10 ng/mL. The repeatability, expressed as Relative Standard Deviation percentage (RSD%) of five determinations, was less than 10% for target compounds except for 2,7-dinitrofluorene, 1,3- and 1,8-dinitropyrene (RSD% &lt; 15%). The limit of detection (LOD) ranged from 12 to 84 pg/mL for most of NPAHs, except for dinitro-pyrenes and nitro-benzo(a)anthracene for which the LOD reached 1.8 ng/mL. The method developed was applied to real samples in order to evaluate the levels of NPAHs in the urban and industrial area of Taranto (South of Italy). The analysis of PM10 samples collected at four industrial and one urban sites, highlighted that in proximity of critical emission source as the biggest European steel plant and under certain weather conditions, combustion processes were the main source of NPAHs in atmosphere

Pentadecanal and pentadecanoic acid coatings reduce biofilm formation of Staphylococcus epidermidis on PDMS

Staphylococcus epidermidis is well known to be one of the major causes of infections related to medical devices, mostly due to its strong capacity to form device-associated biofilms. Nowadays, these infections represent a severe burden to the public health system and the necessity of novel antibacterial strategies for the treatment of these difficult-to-eradicate infections is urgent. The Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 was found to be able to produce an anti-biofilm molecule, the pentadecanal, active against S. epidermidis. In this work, we modified one of the most widely used silicone-based polymers, polydimethylsiloxane (PDMS), by adsorption of pentadecanal and its most promising derivative, pentadecanoic acid, on the PDMS surface. The biofilm formation of S. epidermidis RP62A on both untreated and modified PDMS was performed in a parallel plate flow chamber system, demonstrating the capability of the proposed anti-biofilm coatings to strongly reduce the biofilm formation. Furthermore, drug-release capacity and long-term efficacy (21 days) were also proven for the pentadecanoic acid coating