Recombinant ricin toxin fragments

• US 5,079,163 A
• Filed: 03/12/1987
• Issued: 01/07/1992
• Est. Priority Date: 03/29/1985
• Status: Expired due to Fees

• Current Assignee: Chiron Corporation (Bayer AG), Hewlett-Packard Company (HP Inc.)
• Original Assignee: Cetus Corporation (Bayer AG)
• Inventors: Emerick, Anne W., Houston, L. L., Piatak, Michael Jr.
• Primary Examiner(s): Schwartz, Richard A.
• Application Number:
  US07/025,262
• Time in Patent Office: 1,762 Days
• Field of Search: 435/68, 435/70, 435/172.3, 435/91, 435/320, 435/252.3, 435/320.1, 530/396, 530/377, 530/370, 536/27
• US Class Current: 435/252.3
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/69 Increasing the copy number of the vector
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Ricin B muteins, ricin and ricin precursors having at least one amino acid of at least one galactoside binding site altered to decrease the binding of ricin B to galactosides are claimed. DNA sequences encoding the ricin B muteins, ricin and ricin precursor in which the B chain thereof is the ricin B mutein are claimed. Recombinant expression vectors effective in expressing the DNA sequences of the ricin B muteins, ricin and ricin precursors are claimed. Host cells transformed with the foregoing expression vectors are also claimed. Conjugates comprising binding moieties such as antibodies, hormones, and lymphokines bound to the ricin B mutein and ricin wherein the B chain thereof is the mutein is also claimed.

Claims

1. DNA encoding a ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of Asn255 to either Leu or lie and the substitution of either Tyr248 or His255 to either Gly or Ala.

2. DNA encoding a ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of Tyr248 to Gly, Ala, or Ser.

3. DNA encoding a ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of His251 to Gly, Ala, Asp, Glu, or Lys.

4. DNA encoding a ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of Asn255 to Gly, Ala, Glu, Gln, Asp, or Cys.

5. Recombinant host cells transformed with an expression vector comprising a DNA sequence encoding mutein of ricin B operably linked to control sequences compatible with a recombinant host cell, said mutein being selected from the group consisting of substituting Asn255 to either Leu, Ile, Gly, Ala, Glu, Gln, Asp, or Cys;

Tyr248 or His251 to either Gly, or Ala;

Tyr248 to Gly, Ala, or Ser;

His251 to Gly, Ala, Asp, Glu, or Lys.

6. A replicable, recombinant expression vector effective in expressing the DNA sequence encoding mutein of ricin B, which mutein comprises substitutions selected from the group consisting of Asn255 to either Leu or Ile and the substitution of either Tyr248 or His251 to either Gly, or Ala.

7. A ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of Asn255 to either Leu or Ile and the substitution of either Tyr248 or His255 to either Gly or Ala.

8. A ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of Try248 to Gly, Ala, or Ser.

9. A ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of His251 to Gly, Ala, Asp, Glu, or Lys.

10. A ricin B mutein that exhibits decreased affinity for galactosides, comprising the substitution of Asn255 to Gly, Ala, Blu, Gln, Asp, or Cys.

11. A mutein of ricin B, wherein at least one of the amino acids of positions 37 and 248 is deleted or substituted with a naturally occurring amino acid.