Method for identifying active domains and amino acid residues in polypeptides and hormone variants

A method for identifying at least a first unknown active domain in a region of known amino acid sequence of a naturally occurring parent polypeptide, which parent polypeptide has been cloned and has a preidentified biological activity, said active domain being capable of interacting with a first target when said parent polypeptide is in its native folded form, which interaction is responsible for said biological activity, said method comprising:

• (a) comparing amino acid sequence or polypeptide structure in the region of known amino acid sequence of the parent polypeptide with amino acid sequence or polypeptide structure in a region of known amino acid sequence of an analog polypeptide to the parent polypeptide, said parent polypeptide and said analog both interacting with said first target, resulting in said biological activity, but having different interactions with said first target, or said analog interacting with a different target with which said parent polypeptide also interacts;
• (b) substituting DNA encoding a first analogous polypeptide segment from the analog to said parent polypeptide into DNA encoding substantially the full length parent polypeptide and expressing a first segment-substituted polypeptide;
• (c) contacting said first segment-substituted polypeptide with said first target to determine the interaction, if any, between said first target and said segment-substituted polypeptide;
• (d) repeating steps b) and c) using a second analogous polypeptide segment from an analog to said parent polypeptide to form at least a second segment-substituted polypeptide containing said second analogous polypeptide segment, which is different from said first analogous polypeptide segment;
• and
• (e) comparing the difference, if any, between the activity relative to said first target of said parent polypeptide and said first and second segment-substituted polypeptides as an indication of the location of said first active domain in said parent polypeptide.