Protein CCD at the NKI

2008

 

What's new in ProteinCCD ?

  1. •        31/03/2009 Two more disorder servers are added

  2. •        31/03/2008 A Low Complexity line is added, if the warning occurs.

  3. •        31/03/2009 An Undo button has been anded to clear the latest user action.

  4. •        31/03/2009 The application has now a proper authenticity certificate

  5. •        15/09/2008 The user selections in 'Predictions' (used to be 'Options') are now stored for future sesions.

  6. •        15/09/2008 The button style for Mark start/stop has been changed since some browsers were unhappy with it.

  7. •        15/09/2008 The last sequence used can now be retrieved with the "Last" button.


Will CCD design for me the best constructs for protein expression and crystallization? No. CCD gathers various predictions for your protein and displays the information aligned for easy inspection. CCD allows you then decide where possible constructs should start and stop by simply clcking with the mouse. and will not design the construct automatically.


How does CCD design the PCR oligos? CCD can do that in two ways. The choice can be based on either TM or simply a user choice for length. CCD will not check the oligonucleotides for secondary structure or for cross-hybridization in other sequences and is totally unaware of restriction sites since it was designed to help in LIC cloning.


How do I know that it works? Well, there is no easy way to validate CCD, since the choices that it makes depend on user input. All we can say is that we have used it a lot in our lab at the NKI to make hundreds of constructs, and everybody uses it routinely. CCD will output a vvariety of useful results and its up to you to decide if you want to use them or not and how. The strength of it is that it consolidates the information, and elimintaes the time consuming and error-prone step of 'back-translating' the protein predictions to the DNA sequence.


How can I test ProteinCCD? Just cut and paste the sequence provided in the link under the "Help" menu, and press submit. The translation should appear in the output immediately. The output should indicate a kinase domain at the N-terminus and another domain at the C-terminus. You can inspect the output and choose the domain boundaries you prefer. Then click on 'Get' to get the desired PCR oligos.


How do I get the DNA sequence for my protein? Ideally you have sequenced your starting template and cut-and-paste your actual sequence. You can also get it from any database, just make syre your sequence always starts with ATG and is the valid coding regions of your gene of interest.