I have two independent domain cloned in E.coli and i want to fuse them both just to get a single domain by PCR.
How to get this by PCR?
You could use PCR based methods such as Splicing by Overlap Extension (SOE-PCR); but you need to make sure there are no frameshifts to get a bifunctional enzyme/ chimeric protein.
These articles may help you,
Article Synthetic fusion protein design and applications
Article Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes
Dear Abhilek!
I recommend my previously work. A fused two different protein, but you can use also to domains. Here is the publication:
Article Heterologous Expression and Purification of Recombinant Proa...
If you need more help, I can help you to design the fuse.
Good work!
Overlap extension is the most straightforward method - just make sure to select the right linker. You can find more information about it in my previous works available on researchgate.
You could use PCR-ligationPCR as described here...
Article PCR-ligation-PCR mutagenesis: A protocol for creating gene f...
Hi, I have done this work (linking 2 independent domain) but not by PCR. I can introduce you the method. It was precise but a little long. You shod use appropriate linker to fuse these domains together. Before it , I think it is better to do insilico analyses to predict the structure and function of your fused protein (albeit if you want to express the chimeric gene).
Studies like "In Silico Design of Multimeric HN-F Antigen as a Highly
Immunogenic Peptide Vaccine Against Newcastle Disease Virus" is necessary before your experiments. If you have any question fell free.
Be successful
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning