Saturday, 25 August 2012

GGS LIVE - Protein expression in vertebrate cells

Hello Biofreakers,

I am back BAYBE!!

Today we will cover a topic about expression of protein in vertebrate cells.

Method: Expression of protein X in human osteosarcoma cells (U2OS).

About: Protein expression allows for either purification or study of its cellular function through for example its localisation etc.

What: Expression of RFP-tagged-(Red Fluorescent Protein)-Protein X in U2OS cells to study its localisation.

To express a protein of interest in vertebrate cells we need to have its cDNA sequence. To get the target cDNA we need to clone it from either cDNA library (optionally isolate the cDNA from such library) or from mRNA. When cDNA sequence is ready it is then cloned into a vector that allows expression of that cDNA in vertebrate cells (please see this post GGS LIVE - Making a fusion protein for more details on how to create fusion proteins). Different vectors are available for expression of cDNAs in vertebrate cells. When vector containing cDNA is generated it has to be transfected into cells. This can be achieved by electroporation, chemical reagent such as lipid based transporters and others.

In our study case the U2OS cells will were transfected with lipid based reagent (lipofectamine). In order to detect the protein of interest its expression was monitored by fluorescence microscopy (please see this post for more details GGS LIVE - Immunofluorescence) as shown on Figure below. 


As you can see on the Figure, not transfected cell show very little or no red fluorescence indicating that there is no RFP-Protein X expressed in these cells. On the contrary the cells transfected with the cDNA coding RFP-Protein X fusion show red fluorescence. Moreover, the red fluorescence co-localises with the blue signal that comes from DNA (DNA was stained with DAPI). This suggests that the RFP-Protein X can be mainly found in the nucleus. Therefore, Protein-X is very likely to be a nuclear protein (to test that we could use technique described here GGS LIVE - Cell Fractionation). To confirm that we indeed expressed the RFP-protein X in U2OS cells, protein extract were prepared from these cells after transfection and analysed by immunoblotting (for more details on this technique please see here GGS LIVE - Western Blotting).

As you can see on the Figure below, the immunoblotting showed that RFP-Proiten X fusion can be detected exclusively in the cells that were transfected with the DNA coding for RFP-Protein X (antibody against the RFP was used to detect RFP-Protein X fusion). Detection of Actin protein was used here as loading control showing that the lane NT contains proteins but indeed does not have Protein X.

The biggest issue when expressing the protein of interest in the vertebrate cells is the transfection efficiency. The highest transfection efficiencies are obtained when the specific viruses are used to deliver the DNA into host cells (usually more thatn 90%). Unfortunately, I do not have any experience with this approach, therefore it will not be discussed further here. However, to ensure that as much cells as possible express our protein of interest we can optimise our transfection conditions by testing different ratios of the transfection reagent to DNA. For an example, we can keep the amount of the DNA constant and change the volume of transfection reagent as shown in the table and figure below:

To show you how this was quantified please see the pictures below:

In this case I used Fusion of Green Fluorescent Protein (GFP) with Protein X as my marker. The DNA labelling with DAPI shows the total number of the cells. The number of the cells showing Green Fluorescence by the total number of the cells gives us the transfection efficiency. As you can see when increasing amount of transfection reagent was used the transfection efficiency rised.

Moreover, the protein that we wish to express may not contain tag or be fused to a different type of tag (eg. non-fluorecent tag) that allows for example, to purify and study the biochemistry of the Protein X or to identify other proteins bound to protein X. This can be achieved by immnuprecipitation of the Protein X and analysis of the protein complexes associated with Protein X (for immunoprecipitation thechnique go here Biochemistry Methods - Immunoprecipitation).
As you can see the there is many ways that we could use protein expression as a tool to answer our scientific questions.

I hope you enjoyed it:)



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