cloning into pGEX-4T-1

[BabcockHall]

Hello Everyone,

 

http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/pdfs/GST_gene_fusion_system_handbook.pdf

 

We are trying to understand a plasmid we were sent, and my knowledge of cloning is out of date. The plasmid consists of the phosphatase of interest (of known sequence) cloned into a vector designed to make GST fusion proteins, namely pGEX-4T-1, and there is a site for thrombin cleavage. After the Arg-Gly thrombin site are six nucleotides that would express a serene and a proline residue. The next nucleotides begin a restriction site for EcoRI, GAATTC. The paper we are following indicates that EcoRI and XhoI were the enzymes used to clone the gene of interest.

 

"Collectively, the pGEX vectors provide all three translational reading frames beginning with the EcoRI restriction site (Fig 1.1). pGEX-1λT, pGEX-6P-1, pGEX-4T-1, and pGEX-5X-1 can directly accept and express cDNA inserts isolated from λgt11 libraries." Based upon this passage from the handbook above, I would offer a guess that the phosphatase gene was cloned out of lambda-gt11, but I don't know this with certainty. What I don't know is what lies between the EcoRI site and the beginning of the gene for the phosphatase. Is there any way to make an intelligent guess about this? is there any resource that describes cloning from lambda-gt11 into pGEX-4T-1?

 

We have the mass of the fusion protein, and we would like to compare the predicted versus the theoretical mass to verify the existence of the thrombin cleavage site. Thanks for any help or suggestions.

Edited September 10, 2015 by BabcockHall

[CharonY]

If I understand your question correctly, your insert is cloned in-frame with EcoRI from a lambdagt11 library? In that case, it depends how the phosphatase was cloned into the lambda vector. At most, you will get the 5'UTR of the mRNA, e.g. if it was amplified unidirectionally plus possible linker or adapter sequences. However, with targeted approaches you could start with the start codon, or even just with the part of interest. You would require a bit more info on how the vectors were constructed. Alternatively you could do restriction analyses (if budget is limited) to get an idea or just sequence it, depending on critical your application is.