Protein purification and characterization

1. Protein purification and characterization Exercise #2 In-Silico: Databases & Tools ppc.course@gmail.com

2. Protein Purification & Characterization

3. ? TTTGAAGCGCGTCTGGTGCAGGGCAGCATTCTGAAAAAAGTGCTGGAAGCGCTGAAAGATCTGATTAACGAAGCGTGCTGGGATATTAGCAGCAGCGGCGTGAACCTGCAGAGCatgGATAGCAGCCATGTGAGCCTGGTGCAGCTGACCCTGCGTAGCGAAGGCTTTGATACCTATCGTTGCGATCGTAACCTGGCGatgGGCGTGAACCTGACCAGCatgAGCAAAATTCTGAAATGCGCGGGCAACGAAGATATTATTACCCTGCGTGCGGAAGATAACGCGGATACCCTGGCGCTGGTGTTTGAAGCGCCGAACCAGGAAAAAGTGAGCGATTATGAAatgAAACTGatgGATCTGGATGTGGAACAGCTGGGCATTCCGGAACAGGAATATAGCTGCGTGGTGAAAatgCCGAGCGGCGAATTTGCGCGTATTTGCCGTGATCTGAGCCATATTGGCGATGCGGTGGTGATTAGCTGCGCGAAAGATGGCGTGAAATTTAGCGCGAGCGGCGAACTGGGCAACGGCAACATTAAACTGAGCCAGACCAGCAACGTGGATAAAGAAGAAGAAGCGGTGACCATTGAAatgAACGAACCGGTGCAGCTGACCTTTGCGCTGCGTTATCTGAACTTTTTTACCAAAGCGACCCCGCTGAGCAGCACCGTGACCCTGAGCatgAGCGCGGATGTGCCGCTGGTGGTGGAATATAAAATTGCGGATatgGGCCATCTGAAATATTATCTGGCGCCGAAAATTGAAGATGAAGAAGGCAGC

5. TTTGAAGCGCGTCTGGTGCAGGGCAGCATTCTGAAAAAAGTGCTGGAAGCGCTGAAAGATCTGATTAACGAAGCGTGCTGGGATATTAGCAGCAGCGGCGTGAACCTGCAGAGCatgGATAGCAGCCATGTGAGCCTGGTGCAGCTGACCCTGCGTAGCGAAGGCTTTGATACCTATCGTTGCGATCGTAACCTGGCGatgGGCGTGAACCTGACCAGCatgAGCAAAATTCTGAAATGCGCGGGCAACGAAGATATTATTACCCTGCGTGCGGAAGATAACGCGGATACCCTGGCGCTGGTGTTTGAAGCGCCGAACCAGGAAAAAGTGAGCGATTATGAAatgAAACTGatgGATCTGGATGTGGAACAGCTGGGCATTCCGGAACAGGAATATAGCTGCGTGGTGAAAatgCCGAGCGGCGAATTTGCGCGTATTTGCCGTGATCTGAGCCATATTGGCGATGCGGTGGTGATTAGCTGCGCGAAAGATGGCGTGAAATTTAGCGCGAGCGGCGAACTGGGCAACGGCAACATTAAACTGAGCCAGACCAGCAACGTGGATAAAGAAGAAGAAGCGGTGACCATTGAAatgAACGAACCGGTGCAGCTGACCTTTGCGCTGCGTTATCTGAACTTTTTTACCAAAGCGACCCCGCTGAGCAGCACCGTGACCCTGAGCatgAGCGCGGATGTGCCGCTGGTGGTGGAATATAAAATTGCGGATatgGGCCATCTGAAATATTATCTGGCGCCGAAAATTGAAGATGAAGAAGGCAGCTTTGAAGCGCGTCTGGTGCAGGGCAGCATTCTGAAAAAAGTGCTGGAAGCGCTGAAAGATCTGATTAACGAAGCGTGCTGGGATATTAGCAGCAGCGGCGTGAACCTGCAGAGCatgGATAGCAGCCATGTGAGCCTGGTGCAGCTGACCCTGCGTAGCGAAGGCTTTGATACCTATCGTTGCGATCGTAACCTGGCGatgGGCGTGAACCTGACCAGCatgAGCAAAATTCTGAAATGCGCGGGCAACGAAGATATTATTACCCTGCGTGCGGAAGATAACGCGGATACCCTGGCGCTGGTGTTTGAAGCGCCGAACCAGGAAAAAGTGAGCGATTATGAAatgAAACTGatgGATCTGGATGTGGAACAGCTGGGCATTCCGGAACAGGAATATAGCTGCGTGGTGAAAatgCCGAGCGGCGAATTTGCGCGTATTTGCCGTGATCTGAGCCATATTGGCGATGCGGTGGTGATTAGCTGCGCGAAAGATGGCGTGAAATTTAGCGCGAGCGGCGAACTGGGCAACGGCAACATTAAACTGAGCCAGACCAGCAACGTGGATAAAGAAGAAGAAGCGGTGACCATTGAAatgAACGAACCGGTGCAGCTGACCTTTGCGCTGCGTTATCTGAACTTTTTTACCAAAGCGACCCCGCTGAGCAGCACCGTGACCCTGAGCatgAGCGCGGATGTGCCGCTGGTGGTGGAATATAAAATTGCGGATatgGGCCATCTGAAATATTATCTGGCGCCGAAAATTGAAGATGAAGAAGGCAGC

6. Sequence identification • Should we work with DNA? • How can we identify the amino acid sequence behind the DNA sequence? • What should we confirm before in-silico translation of the amino acid sequence?

7. http://www.expasy.org

10. PASTE YOUR SEQUENCE HERE (FASTA FORMAT)

15. Basic Local Alignment Search ToolBLAST blastp Protein blast blastn Nucleotide blast

19. <50 is unreliable 10-5 is unreliable >25 is good news

21. what additional information we need gather before a protein purification scheme?

23. ProtParam Physico-chemical parameters of a protein sequence Why is the pI therotical?

24. interPro is a database of protein families, domains, regions, repeats and sites in which identifiable features found in known proteins can be applied to new protein sequences.

25. InterProScan http://www.ebi.ac.uk/Tools/InterProScan/

26. Secondary Structure prediction

27. CDD – Crystallization Construct Designer http://xtal.nki.nl/ccd/start_ccd.html

28. Post-translational modification prediction http://www.cbs.dtu.dk/services/SignalP/ Why we should identify these signals prior a purification? Clevage site Signal sequence

29. DiANNA 1.1 web server

31. How should we adapt our purification scheme when predicting disulfide bond(s)? • What potential effects such disulfide bond(s) have on our protein and its purification scheme?

37. Homology modeling

38. TAKES A LOT OF TIME!

39. Another excellent way to find bioinformatics tool - GOOGLE

40. Data bases