Directed evolution of disulfide-bonded micro-proteins

• US 20060084113A1
• Filed: 07/31/2002
• Published: 04/20/2006
• Est. Priority Date: 09/01/1989
• Status: Active Grant

• Current Assignee: Dyax Corporation (Shire plc)
• Original Assignee: Dyax Corporation (Shire plc)
• Inventors: Ladner, Robert Charles, Ley, Arthur Charles, Kent, Rachel Baribault, Markland, William, Roberts, Bruce Lindsay, Guterman, Sonia Kosow
• Granted Patent: US 7,413,537 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

In order to obtain a novel binding protein against a chosen target, DNA molecules, each encoding a protein comprising one of a family of similar potential binding domains and a structural signal calling for the display of the protein on the outer surface of a chosen bacterial cell, bacterial spore or phage (genetic package) are introduced into a genetic package. The protein is expressed and the potential binding domain is displayed on the outer surface of the package. The cells or viruses bearing the binding domains which recognize the target molecule are isolated and amplified. The successful binding domains are then characterized. One or more of these successful binding domains is used as a model for the design of a new family of potential binding domains, and the process is repeated until a novel binding domain having a desired affinity for the target molecule is obtained. In one embodiment, the first family of potential binding domains is related to bovine pancreatic trypsin inhibitor, the genetic package is M13 phage, and the protein includes the outer surface transport signal of the M13 gene III protein.

Claims

1-60. -60. (canceled)

61. A library of chimeric proteins, each chimeric protein comprising:

a) a mini-protein between about eight and about forty amino acids long, wherein the mini-protein has a single disulfide bond formed by a pair of invariant cysteines and

b) at least a portion of an outer surface protein of a genetic package,

wherein the chimeric protein is displayed on the outer surface of the genetic package.

137. The library of claim 61, wherein the span of the disulfide bond is at least two amino acid residues and no more than about nine amino acid residues.

138. The library of claim 61, wherein the span of the disulfide bond is 3 amino acids.

139. The library of claim 61, wherein the span of the disulfide bond is 4 amino acids.

140. The library of claim 61, wherein the span of the disulfide bond is 5 amino acids.

141. The library of claim 61, wherein the span of the disulfide bond is 6 amino acids.

142. The library of claim 61, wherein the span of the disulfide bond is 7-9 amino acids.

143. The library of claim 61, wherein at least one of the amino acids in the span of the disulfide bond is Gly in at least some of the mini-proteins in the library.

144. The library of claim 61, wherein at least one of the amino acids in the span of the disulfide bond is Gly in all of the mini-proteins in the library.

145. The library of claim 61, wherein the span of the disulfide bond comprises at least one main-chain geometry constraining amino acid in at least some of the mini-proteins in the library.

146. The library of claim 145, wherein the at least one main-chain geometry constraining amino acid is proline, valine, or isoleucine.

147. The library of claim 146, wherein the at least one main-chain geometry constraining amino acid is proline.

148. The library of claim 61, wherein the span of the disulfide bond comprises at least one main-chain geometry constraining amino acid in all of the mini-proteins in the library.

149. The library of claim 148, wherein the at least one main-chain geometry constraining amino acid is proline, valine, or isoleucine.

150. The library of claim 149, wherein the main-chain geometry constraining amino acid is proline.

151. The library of claim 61, wherein the mini-protein comprises an amino acid sequence of 20 or fewer amino acids.

152. The library of claim 61, wherein the amino acids of the mini-protein sequence, other than the cysteines, are variable.

153. The library of claim 61, wherein each variable amino acid can be substituted by any one of at least eight different amino acids.

154. The library of claim 61, wherein the span of the disulfide bond comprises one or more variable amino acids.

155. The library of claim 61, wherein all amino acids included in the span of the disulfide bond, other than any main-chain geometry constraining amino acids, are variable.

156. The library of claim 61, where the mini-protein comprises the sequence X₁—

C—

X₂—

X₃—

X₄—

X₅—

C—

X₆

wherein the cysteines (Cs) form a disulfide bond, and wherein X₁ through X₆ are amino acids that are varied independently of each other.

157. The library of claim 61 wherein the disulfide bond bridges a sequence of amino acids that collectively assume a hairpin secondary structure under affinity separation conditions.

158. The library of claim 157, wherein the hairpin secondary structure is a) an alpha helix, a turn, and a beta strand;

b) an alpha helix, a turn, and an alpha helix;

or c) a beta strand, a turn, and a beta strand.

159. The library of claim 61, wherein at least one amino acid that is varied in said mini-protein is encoded by a variegated codon chosen from NNT, NNG, RNG, RMG, VNT, RRS, and SNT.

160. The library of claim 61, wherein none of the amino acids that are varied in said mini-protein is encoded by a variegated codon chosen from NNN, NNK and NNS.

161. The library of claim 61, wherein the mini-protein is directly fused to at least a portion of the outer surface protein.

162. The library of claim 61, wherein the genetic package is a filamentous phage.

163. The library of claim 162, wherein the mini-protein is encoded by a phagemid.

164. The library of claim 162, wherein the outer surface protein is the major coat protein of a filamentous phage.

165. The library of claim 162, wherein the outer surface protein is the gene III protein of a filamentous phage.

166. The library of claim 61, wherein the genetic package is a bacterial cell.

167. The library of claim 166, wherein the bacterial cell is Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, or Escherichia coli.

168. The library of claim 61, wherein the genetic package is a bacterial spore.

169. The library of claim 61, wherein the chimeric protein binds a target.

170. The library of claim 169, wherein the target is a serine protease.

171. The library of claim 170, wherein the serine protease is human neutrophil elastase.

172. A process for identifying proteins with a desired binding activity against a target which comprises:

a) screening a library of chimeric proteins according to claim 61;

and

b) identifying the chimeric protein.

173. A chimeric protein expressed by a library according to claim 61.

174. A mixture of nucleic acids, wherein the mixture encodes the library of claim 61.

175. The mixture of claim 174, wherein each nucleic acid is cloned in an expression vector.

62-136. -136. (canceled)