Protein Expression Prokaryotic cells - B. subtilis PHY US417

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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pAF3

Hichem Chouayekh, Laboratoire de Microorganismes et de Biomoléc

Upstream tips	Protocol tips	Downstream tips
	B. subtilis was transformed according to the method of Anagnostopoulos and Spizizen (1961) with some modifications. To obtain naturally competent cells, B. subtilis 168 was grown in the Spizizen minimal medium (SMM): 80 mM K2HPO4, 45 mM KH2PO4, 15 mM (NH4)2SO4 and 3.8 mM Na3-citrate, supplemented with 5 mM MgSO4, 5 g l-1 glucose, 0.5 g l-1 tryptophan and 0.1 g l-1 casaminohydrolysate. For efficient DNA uptake of pAF3 and pMSP3535 (negative control) by B. subtilis, the plasmid DNA (1 μg) was linearized by NsiI and self-ligated in vitro to generate multimeric plasmidic forms. After dilution of competent cells (10-1) in SMM containing 20 mM MgCl2 and 5 g l-1 glucose, pAF3 or pMSP3535 plasmid DNA multimers were added, and the samples were incubated for 20 min at 37°C. Transformation mixtures were subsequently spread on LB agar containing erythromycin (5 μg ml-1).	B. subtilis transformants were screened for the ability to produce phytase activity on LB agar supplemented with phytic acid (3 mM) by using the well-known two step counterstaining treatment (Bae et al. 1999). Colonies surrounded by clear zones were tested by PCR to confirm the presence of the phy US417 gene.

Protocol tips
B. subtilis was transformed according to the method of Anagnostopoulos and Spizizen (1961) with some modifications. To obtain naturally competent cells, B. subtilis 168 was grown in the Spizizen minimal medium (SMM): 80 mM K2HPO4, 45 mM KH2PO4, 15 mM (NH4)2SO4 and 3.8 mM Na3-citrate, supplemented with 5 mM MgSO4, 5 g l-1 glucose, 0.5 g l-1 tryptophan and 0.1 g l-1 casaminohydrolysate. For efficient DNA uptake of pAF3 and pMSP3535 (negative control) by B. subtilis, the plasmid DNA (1 μg) was linearized by NsiI and self-ligated in vitro to generate multimeric plasmidic forms. After dilution of competent cells (10-1) in SMM containing 20 mM MgCl2 and 5 g l-1 glucose, pAF3 or pMSP3535 plasmid DNA multimers were added, and the samples were incubated for 20 min at 37°C. Transformation mixtures were subsequently spread on LB agar containing erythromycin (5 μg ml-1).
Downstream tips
B. subtilis transformants were screened for the ability to produce phytase activity on LB agar supplemented with phytic acid (3 mM) by using the well-known two step counterstaining treatment (Bae et al. 1999). Colonies surrounded by clear zones were tested by PCR to confirm the presence of the phy US417 gene.

Publication protocol 1 Relevant paper

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