There is no better than the other cell line to express a surface protein.

CHO, Hela, COS 7, COP5 or HEK (293 or 293T) are equivalent: only the growth and size differentiate them. Which may interfere with the transfection ability. Be chosen transfection mode according to the cell used.

  For example: HEK and COP5 for transfection by electroporation. Hela and CHO for chemical transfection such as PEI.

But most important for the size of the plasmid: small (

There is no better than the other cell line to express a surface protein.

CHO, Hela, COS 7, COP5 or HEK (293 or 293T) are equivalent: only the growth and size differentiate them. Which may interfere with the transfection ability. Be chosen transfection mode according to the cell used.

  For example: HEK and COP5 for transfection by electroporation. Hela and CHO for chemical transfection such as PEI.

But most important for the size of the plasmid: small (

It would help if you described what you are trying to achieve. Do you seek the highest possible expression because you wish to purify it or create a membrane preparations for binding says etc, or are you looking to build a functional assay? If you need relatively few cells then transient expression systems could be used to overcome any issues you may have had with stable expression. Does the GPCR need to be expressed in adherent or suspension cultures. It would also help if you name the GPCR or at least its ligand. Sometimes poor GPCR expression can be caused by culture conditions e.g. 5HT receptors internalising due to 5HT in serum containing media. The low mwt in SDS gel does not sound encouraging. How low? Have you confirmed the sequence in your construct is correct? 

Thank you Jean-Jacques and Jim. A little clarifications from you observations. I am using lipofection to transfect. My vector is pcDNA 3.1 and the gene is about 1578 bp giving a total of about 7000bp.

As for the other questions: I intend to use this cells mainly for protein purification and as for whether they have to expressed in adherent or suspension cultures, I will be grateful if you can give me some advice on that (I am very new to cell culture).

And as for the GPCR name, its is novel and we seek to deorphanize it using the Galpha 16 promiscuous protein. For the ligands, we have no idea as the close homologs are not annotated.

The molecular weight is about 60 kDa but we detected 2 proteins with sizes 49 and 38 kDa. We are still waiting for mass spectrometry results.

Hope this make the question clearer.

There, in your project, two main questions:

1. express the GPCR at a good level.

2. recovering the protein in its native conformation to use in assays, immunological likely.

First: pcDNA3.1 may be used with the PEI rather than lipofectamine or equivalent. Otherwise, due to the relatively small size of the gene, the association with a very small vector, would use the elecroporation which is much effective.And performance is a key point for the study of GPCRs.

For the second: the recovery of the GPCR is with cocktails of very specific detergents (and for many patented). I encourage you to contact the Calixar company, which specializes in field.

OK Robert I will try and offer advice on each option available as related to your different objectives. Firstly you should know that if you are trying to purify a GPCR in any quantity and you are new to cell culture, you are right in the deep end. This would be true for an 'easy' to express GPCR, never mind a novel one! If you need anything more than 100ug I would look to baculoviral or microbial expression and a lot of help from experts. If you just need enough in semi-purified form to perform binding assays then suspension cultures of CHO cells up to about 10 litres may well provide enough material. In general adherent cultures are more difficult to work with at scale, but this depends on facilities and expertise available. Some GPCR's express better in adherent cells, and most people can probably scale up to say 8 x 6360cm2 (10 stacks) within standard incubators / cabinets etc. If you have facility to generate large amounts of DNA, then you can consider transient as well as stable expression. To maximise transcription with pcDNA3.1 you will need a host that expresses Large T antigen to take advantage of the SV40 origin of replication and see episomal replication within 48-72 hrs transient expression. The latter is not always essential, but it usually makes a big difference. HEK cells that express T ag are commercially available but I'm not sure about CHO. If you need transient expression in the latter then you may need to use a different vector. NB: You would as Jean-Jacques suggests need to use PEI etc as Lipofectamine would prove expensive large scale!

For stable expression you have a wider choice of hosts and culture methods. Moreover the higher transcription rates that can be obtained within transient expression does not always lead to higher protein expression of GPCRs as it would with say soluble secreted proteins. It's not unusual to get stable GPCR expression close to or equal to what can be achieved transiently. The exceptions to this are GPCRs whose long term expression is actively minimised by host cells. To predispose selection of clones that express GPCRs at the highest levels, use a bicistronic vector where the IRES element is deliberately weakened.

If you wish to de-orphanise, then functional assays with prospective ligands could well be the way ahead. This reduces the number of cells required and also the level of expression needed. 

I would suggest that as well as CHO and HEK etc you consider hosts with a similar phenotype to the cells your GPCR was isolated from. This could be vital for expression and /or function. If you have trouble detecting either of the latter try a) a period in culture after the cells have been washed free of growth factors / serum etc, b) rebuilding the construct with the ORF protein sequence made with a different nucleotide sequence.