Protein Expression Prokaryotic cells - Brevibacillus choshinensis SP3 α-amylase

Protein expression refers to the techniques in which a protein of interest is synthesized, modified or regulated in cells. The blueprints for proteins are stored in DNA which is then transcribed to produce messenger RNA (mRNA). mRNA is then translated into protein. In prokaryotes, this process of mRNA translation occurs simultaneously with mRNA transcription. In eukaryotes, these two processes occur at separate times and in separate cellular regions (transcription in nucleus and translation in the cytoplasm). Recombinant protein expression utilizes cellular machinery to generate proteins, instead of chemical synthesis of proteins as it is very complex. Proteins produced from such DNA templates are called recombinant proteins and DNA templates are simple to construct. Recombinant protein expression involves transfecting cells with a DNA vector that contains the template. The cultured cells can then transcribe and translate the desired protein. The cells can be lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic protein expression systems are widely used. The selection of the system depends on the type of protein, the requirements for functional activity and the desired yield. These expression systems include mammalian, insect, yeast, bacterial, algal and cell-free. Each of these has pros and cons. Mammalian expression systems can be used for transient or stable expression, with ultra high-yield protein expression. However, high yields are only possible in suspension cultures and more demanding culture conditions. Insect cultures are the same as mammalian, except that they can be used as both static and suspension cultures. These cultures also have demanding culture conditions and may also be time-consuming. Yeast cultures can produce eukaryotic proteins and are scalable, with minimum culture requirements. Yeast cultures may require growth culture optimization. Bacterial cultures are simple, scalable and low cost, but these may require protein-specific optimization and are not suitable for all mammalian proteins. Algal cultures are optimized for robust selection and expression, but these are less developed than other host platforms. Cell-free systems are open, free of any unnatural compounds, fast and simple. This system is, however, not optimal for scaling up.

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Found 1 matching solution for this experiment

SEC-GFP-pHis1522

Manuele Martinelli, Novartis Vaccines and Diagnostics, Via Fiore

Upstream tips
Brevibacillus competent cells were prepared according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan).
Protocol tips
All the expression vectors were transformed into Brevibacillus choshinensis SP3 using the tris-polyethylene glycol method [4] as described by Takara Bio manual using 0,5-2 μg of total DNA. 10 μg/mL of neomycin and 20 μg/mL of tetracycline were respectively used to transform the plasmid constricts. To screen for protein production freshly transformed cells were grown overnight in 10 mL of TM medium containing 10 μg/mL of neomycin (pNI-His and pNC-His vectors) or 20 μg/mL of tetracycline (pHis1522 and SEC-pHis1522 vectors) at 30°C, 150 rpm. Pre-inoculums were inoculated 1:100 in 50 mL of the same media and grown at 150 rpm at 25°C, 30°C and 37°C.
Downstream tips
Recombinant expression of the selected proteins under transcriptional control of the xylose-inducible promoter, were induced by the addition of xylose at different concentrations (0,5-1-2%) at OD600 about 1,5. Intracellular GFP, α-amylase and TcdA-GT samples were withdrawn at different growth phases, and subjected to Western blotting analysis, GFP fluorescence determination and α-amylase activity assay as described below.
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