I am to clone a chimeric protein gene sequence using the PFastBac HT C vector. However, I want to insert a signal peptide which will make my expressed protein to target the endoplasmic reticulum. I am worried about a few things?
1. Is this even possible?
2. How can I perform this?
3. At what position on my vector am I to insert the signal peptide sequence?
You can insert the signal sequence by overlapping PCR strategy with 20 - 25 bp overlaps between your signal sequence and gene sequence. However, you need to know where exactly it should be in your expressed protein and it should not interfere with the function of the protein. I normally suggest inserting it at N-terminal or C-terminal end. You need to carefully study the structure of your protein and how signal peptide works. Then you can devise a plan. More details are needed to understand your problem.
please see here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1576044/
Usually signal peptide is inserted at the amino-terminus of your sequence. You could clone the whole gene (coding for signal peptide+your protein) or clone the signal peptide gene alone before. You can play with the available restriction sites. Its recommendable to have a restriction site overlapping with the end of the signal peptide gene to be able to clone different genes with the same signal peptide without introducing additional residues at the joints.
You can also introduce the signal peptide gene by mutagenesis methods based on plasmid amplification like quick change mutagenesis or Kunkel mutagenesis, but there is certain probability of introducing undesired mutations at the vector backbone. Ideally you would need to sequence the whole vector after that. Thats why I prefer cloning using restriction sites, to be sure that the rest of the vector is not changed.
You need to select a suitable signal peptide that has been used to secrete recombinant proteins by your host cells.You should check in silico that in your host cells the signal peptidase is able to cleave the resulting polypeptide just between the signal peptide and the beginning of your protein. This can be predicted by several available softwares. If there are problems to cut, you can add or delete a few aminoacids at the N-terminus of your protein (depending on the functional importance of taht region) until you design a sequence that can be properly cleaved by the signal peptidase.
Thank you very much for your elaborate answer, Getrudis. Please can you recommend one such prediction softwares for me?
Just google signal peptidase cut prediction or something like that. Several options will appear.
Thank you all. Dr Rojas and Dr Budida, I intend to insert the signal peptide sequence at the N-terminal of my protein's gene sequence. However, is the signal protein definitely going to be cleaved off after trafficking my protein with the cell? I intend to target the ER.
Make sure that how the cleavage functions in proteins from which the signal sequence is obtained and try to include the cleavage site along with the signal sequence.
Goodluck
Normally proteins go to the ER because of the presence of a signal peptide, then they are released from the SP and are secreted or anchored to the membrane if they have a transmembrane domain. Do you want your protein to stay at the ER instead of being secreted? In that case you should add an ER-retention signal like KDEL. Search for articles dealing with ER-retention signals, for instance for intrabody retention at the ER.
Hi Dr. Rojas. No, I want my protein to be secreted. The signal peptide I want to use is about 100aa in length and the prediction software showed that the signal peptide's cleavage site is around 70aa. That suggests that the remaining 30aa will be attached to my protein and I will not want that because it might affect the properties of my protein.
This SP is longer than usual. Is this the SP attached to your protein in nature or a SP commonly used for expression of recombinant proteins in your host? I normally use smaller SP and cleavage occurs just at the end, sometimes levaing a few additional residues due to the cloning strategy.
Okay. But is 24aa also longer than usual? I am considering an alternative.
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