pET restriction site advice

[anteloop]

Hello,

I'm a student and new to the forum and wondered if anyone could give me some advice.

I have a glycoprotein fragment which I cloned into a retroviral expression vector. This has expressed well and the proteins fine for binding studies etc. The problem I have is that the fragment is N glycosylated (only 1 site) and when it runs on a gel it runs as a tight doublet. The top band is the glycosylated protein.

In the long-term I hoped to maybe set up some crystal trials using the protein as it expresses so well but I've been told glycosylation is a big no no.

Looking at the literature, groups who have crystalised the protein have used either baculoviral or bacterial vectors which I assume was partly to get over the glycosylation. The fragment I have was a bit of a nightmare to clone so I'm reluctant to go back and re-design primers. I have it in the Topo 2.1 TA vector and the fragment has:

AgeI and PacI site

XhoI and BamHI site

We have the pET 28a and pET 48b vectors in the lab. On the multiple cloning sites there is a BamHI and XhoI site so I thought maybe I could double digest my fragment out of Topo and then ligate into double digested pET vector for expression in E-coli?

My XhoI and BamHI sites are back to front and hope this question doesn't sound too silly but, does it matter which way round my fragment goes into the pET vector?

Which pET vector would be most recommended?

Are there any other bacterial expression vectors which may be complementary to my restriction sites?

Any advice would be much appreciated!

[clevermizo]

My XhoI and BamHI sites are back to front and hope this question doesn't sound too silly but, does it matter which way round my fragment goes into the pET vector?

I'm pretty sure it does. You want the gene cloned in so that the T7 promoter is 5' to the gene. The T7 promoter has a directionality to it, and it should be 5' of the MCS. If you clone it in the opposite way, it may not express or you'll get something different than what you'd like.

Now from what I know of TOPO TA, the BamHI site is 5' of the XhoI site which is 3' to the gene. Could you comment on the actual directionality of your fragment with respect to the restriction sites? TOPO TA can clone PCR products in either way. Is it:

1) BamHI ........ GENE -->>>>> ....... XhoI

or

2) BamHI ...... <<<<<<-------- GENE ....... XhoI ?


If it's situation 1), you can't clone it in the right orientation into the pET MCS. But what about BamHI/ApaI? BamHI and ApaI seem to have the same directionality in both pcr2.1 and the pET MCSs. ApaI digest work just fine, but there's a trick:

1. You have to transform your vectors into dam/dcm neg E. coli because ApaI is dam/dcm methylation sensitive.
2. ApaI digests best at RT. So add ApaI enzyme, digest for awhile then add BamHI and kick it up to 37 for the rest of the digest.


In terms of recommending pET vectors I can't say because I haven't personally used them.

[anteloop]

Hi,

Thanks for the useful advice

It turns out my Bam and Xho sites are in the correct orientation for the pET vector so going to go ahead and try these out.

[clevermizo]

That sounds like a good idea. Happy to help.