Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii.

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h-1 - Relative Fluorescence Unit h-1 ), in comparison to batch cultivation (174.0 RFU h-1 ) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L-1 .

Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry.

The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

Agrobacterium tumefaciens-mediated transient transformation of Arabidopsis thaliana leaves.

Transient assays provide a convenient alternative to stable transformation. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Nevertheless, at difference of tobacco and other plant species, Arabidopsis thaliana remains recalcitrant to routine transient assays. In this chapter, we describe a transient expression assay using simple infiltration of intact Arabidopsis leaves with Agrobacterium tumefaciens carrying a plasmid expressing a reporter fluorescent protein. In this protocol, Agrobacterium aggressiveness was increased by a prolonged treatment in an induction medium deficient in nutrients and containing acetosyringone. Besides, Arabidopsis plants were cultivated in intermediate photoperiod (12 h light-12 h dark) to promote leaf growth.

Using hyper as a molecular probe to visualize hydrogen peroxide in living plant cells: a method with virtually unlimited potential in plant biology.

Reactive oxygen species (ROS) are highly reactive reduced oxygen molecules that play a myriad of roles in animal and plant cells. In plant cells, the production of ROS occurs as a result of aerobic metabolism during respiration and photosynthesis. Therefore mitochondria, chloroplasts, and peroxisomes constitute an important source of ROS. However, they can be produced in response to many physiological stimuli such as pathogen attack, hormone signaling, abiotic stresses, or during cell wall organization and plant morphogenesis. Monitoring ROS in plant cells has been limited to biochemical assays and use of fluorescent probes, however, the irreversible oxidation of the fluorescent dyes make it impossible to visualize dynamic changes of ROS. Hyper is a recently developed live cell probe for H2O2 and consists of a circularly permutated YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide (H2O2) sensor OxyR rendering it a H2O2 specific ratiometric, and therefore quantitative probe. Herein, we describe a protocol for using Hyper as a dynamic probe for H2O2 in Arabidopsis with virtually unlimited potential to detect H2O2 throughout the plant and under a broad range of developmental and environmental conditions.

Intracellular release of recombinant green fluorescent protein (gfp(uv)) from Escherichia coli.

The recombinant green fluorescent protein (gfp(uv)) was expressed by Escherichia coli DH5-alpha cells transformed with the plasmid pGFPuv. The gfp(uv) was selectively permeabilized from the cells in buffer solution (25 mM Tris-HCl, pH 8.0), after freezing (-70 degrees C for 15 h), by four freeze (-20 degrees C)/thaw cycles interlaid by sonication. The average content of released gfp(uv) (experiment 2) was 7.76, 34.58, 39.38, 12.90, and 5.38%, for the initial freezing (-70 degrees C) and the first, second, third and fourth freeze/thaw cycles, respectively. Superfusion on freezing was observed between -11 degrees C and -14 degrees C, after which it reached -20 degrees C at 0.83 degrees C/min.

Selective permeation and organic extraction of recombinant green fluorescent protein (gfpuv) from Escherichia coli.

BACKGROUND: Transformed cells of Escherichia coli DH5-alpha with pGFPuv, induced by IPTG (isopropyl-beta-d-thiogalactopyranoside), express the green fluorescent protein (gfpuv) during growth phases. E. coli subjected to the combination of selective permeation by freezing/thawing/sonication cycles followed by the three-phase partitioning extraction (TPP) method were compared to the direct application of TPP to the same culture of E. coli on releasing gfpuv from the over-expressing cells. MATERIAL AND METHODS: Cultures (37 degrees C/100 rpm/ 24 h; mu = 0.99 h(-1)-1.10 h(-1)) of transformed (pGFP) Escherichia coli DH5-alpha, expressing the green fluorescent protein (gfpuv, absorbance at 394 nm and emission at 509 nm) were sonicated in successive intervals of sonication (25 vibrations/pulse) to determine the maximum amount of gfpuv released from the cells. For selective permeation, the transformed previously frozen (-75 degrees C) cells were subjected to three freeze/thaw (-20 degrees C/ 0.83 degrees C/min) cycles interlaid by sonication (3 pulses/6 seconds/25 vibrations). The intracellular permeate with gfpuv in extraction buffer (TE) solution (25 mM Tris-HCl, pH 8.0, 1 mM beta-mercaptoethanol beta-ME, 0.1 mM PMSF) was subjected to the three-phase partitioning (TPP) method with t-butanol and 1.6 M ammonium sulfate. Sonication efficiency was verified on the application to the cells previously treated by the TPP method. The intra-cell releases were mixed and eluted through methyl HIC column with a buffer solution (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). RESULTS: The sonication maximum released amount obtained from the cells was 327.67 microg gfpuv/mL (20.73 microg gfpuv/mg total proteins-BSA), after 9 min of treatment. Through the selective permeation by three repeated freezing/thawing/sonication cycles applied to the cells, a close content of 241.19 microg gfpuv/mL (29.74 microg gfpuv/mg BSA) was obtained. The specific mass range of gfpuv released from the same cultures, by the three-phase partitioning (TPP) method, in relation to total proteins, was higher, between 107.28 microg/mg and 135.10 microg/mg. CONCLUSIONS: The selective permeation of gfpuv by freezing/thawing/sonication followed by TPP separation method was equivalent to the amount of gfpuv extracted from the cells directly by TPP; although selective permeation extracts showed better elution through the HIC column.

Nuclear pore ion channel activity in live syncytial nuclei.

Nuclear pore complexes (NPCs) are important nanochannels for the control of gene activity and expression. Most of our knowledge of NPC function has been derived from isolated nuclei and permeabilized cells in cell lysates/extracts. Since recent patch-clamp work has challenged the dogma that NPCs are freely permeable to small particles, a preparation of isolated living nuclei in their native liquid environment was sought and found: the syncytial nuclei in the water of the coconut Cocos nucifera. These nuclei have all properties of NPC-mediated macromolecular transport (MMT) and express foreign green fluorescent protein (GFP) plasmids. They display chromatin movement, are created by particle aggregation or by division, can grow by throwing filaments to catch material, etc. This study shows, for the first time, that living NPCs engaged in MMT do not transport physiological ions - a phenomenon that explains observations of nucleocytoplasmic ion gradients. Since coconuts are inexpensive (less than US$1/nut per litre), this robust preparation may contribute to our understanding of NPCs and cell nucleus and to the development of biotechnologies for the production of DNA, RNA and proteins.