Protein Expression Prokaryotic cells - E. coli SnRK2 A. thaliana

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.

1 Matching solution
Start a discussion

Start discussion

No discussions found

Start your discussion

Share your thoughts or question with experts in your field

Start a discussion

Found 1 matching solution for this experiment

pIVEX2.4c-SnRK2.1

Yi Wan, Microbiology Institute of Shaanxi, Shaanxi Academy of Sc

Upstream tips	Protocol tips	Downstream tips
	The SnRK2.1 protein was expressed in a cell-free expression system based on an E. coli ribosomal extract (S30 extract). S30 extract was prepared as previously reported [18] with some modifications. Briefly, in order to bypass the exogenous addition of commercial creatine kinase (CK) and T7 RNA polymerase (T7 RNAP) into the cell-free reaction mixtures, two types of S30 extracts (CK-T7 extract and BL21 extract) were prepared. The CK-T7 extract was prepared from the E. coli strain BL21 Star (DE3) (Life Technologies) harboring a CK and T7 RNAP expression vector pETDuet-CK-T7 (Figure 1). CK and T7 RNAP were expressed during the cultivation of E. coli via IPTG induction (1.0 mM) at 0.5 OD600.

Protocol tips
The SnRK2.1 protein was expressed in a cell-free expression system based on an E. coli ribosomal extract (S30 extract). S30 extract was prepared as previously reported [18] with some modifications. Briefly, in order to bypass the exogenous addition of commercial creatine kinase (CK) and T7 RNA polymerase (T7 RNAP) into the cell-free reaction mixtures, two types of S30 extracts (CK-T7 extract and BL21 extract) were prepared. The CK-T7 extract was prepared from the E. coli strain BL21 Star (DE3) (Life Technologies) harboring a CK and T7 RNAP expression vector pETDuet-CK-T7 (Figure 1). CK and T7 RNAP were expressed during the cultivation of E. coli via IPTG induction (1.0 mM) at 0.5 OD600.

Protocol tips

The SnRK2.1 protein was expressed in a cell-free expression system based on an E. coli ribosomal extract (S30 extract). S30 extract was prepared as previously reported [18] with some modifications. Briefly, in order to bypass the exogenous addition of commercial creatine kinase (CK) and T7 RNA polymerase (T7 RNAP) into the cell-free reaction mixtures, two types of S30 extracts (CK-T7 extract and BL21 extract) were prepared. The CK-T7 extract was prepared from the E. coli strain BL21 Star (DE3) (Life Technologies) harboring a CK and T7 RNAP expression vector pETDuet-CK-T7 (Figure 1). CK and T7 RNAP were expressed during the cultivation of E. coli via IPTG induction (1.0 mM) at 0.5 OD600.

Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment