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help with protein localization study using GFP - (Aug/14/2010 )

Hi,

I wish to determine localization of a protein in E.coli using gfp. I have never worked with gfp and need some help. I have a few questions

1. I've read that gfp has to be fused with the protein of intrest inframe. so does that mean that there must be no nucleotide gap between the protein coding region and the gfp fusion? if there must be no gap, plzz let me know what to do since I dont have restriction sites right at the end or beginning of coding sequence
2. do I have to fuse my entire protein of intrest or will it be fine if i fuse just a part of my protein of intrest...something like a localization signal along with the gfp? I kindly request you to support the answer with any citation if possible.

Thanks in advance

Ajay

-ajay.s-

Basically you need to make a plasmid containing the GFP and your gene of interest - it will be best if you obtain a GFP plasmid and subclone your gene into it. The plasmid your gene is currently in should have some restriction sites in the mulitple cloning site that will be suitable. If not you can ligate on adapters containing the appropriate sites.

You can have a few nucleotides between the GFP and you gene, but you don't want it to be too long.

You can fuse the whole protein or only part of the protein if you wish, however in either case you should determine whether or not the protein is functioning as it is expected to. Part of the problem with tags such as GFP is that they are massive (26.9 kDa) and as such can easily block normal function... You can consider using a smaller tag and doing something like immunofluoresence.

-bob1-

Thanks a ton..

But, can you suggest any research articles, if you came across, which says that partial protein can be used. I only intend to know where the protein is localizing. I dont intend to know if it is functioning there.

Also, All the articles I came across says that GFP has to be cloned inframe without any gap in between. Can you please suggest me any supporting articles to verify if it can be cloned with a small gap.

-ajay.s-

A quick google scholar search found this paper:

Characterization and localization of the even-skipped protein of Drosophila. M Frasch, T Hoey, C Rushlow, H Doyle, M - The EMBO , 1987.

Localisation and function are usually quite initmately tied in - if the protein is not functional, how do you know that it is localised correctly?

The GFP must be in-frame as otherwise either the GFP or the protein of interest will not be expressed properly. I don't use GFP myself, but I do use some shorter tags, and often have short spacers (6 or 9 bases to keep them in-frame) between them and the gene of interest.

-bob1-

Are you looking to see the localization of an E. coli protein in a host cell, or localization of a protein within E. coli?

You can fuse the whole protein or truncations. I don't have references at my disposal at this moment, but I work with a type III secretion system, and some of the literature I've read regarding T3SS translocated effectors have used fusions, either full length or truncated, to reporters. In-frame simply means the codons have to remain intact for GFP relative to the start codon of your protein of interest. For example, ATG...TGCATG where the second ATG is the start of GFP and the TGC is a codon of your protein of interest. ATG...ACATG or ATG...AATG would not work, because the start of GFP is out of frame with the start of the protein of interest. Also keep in mind that you don't want to include the stop codon of your gene of interest before GFP, otherwise GFP won't be included in translation. In regards to cloning GFP immediately after your gene of interest, that is not necessary. It's common to put a linker between the gene of interest and the GFP. The linker may include restriction sites used for cloning the gene of interest to GFP. Keep in mind that you want the DNA of the linker to be a multiple of 3 to keep the GFP in-frame with the gene of interest.

-fishdoc-

thanks a lot...but do I have to clone the gene along with its promoter for proper localization or just cloning the gene would be fine?

-ajay.s-