Minimization of a polypeptide hormone

Science

Volumn 270, Issue 5242, 1995, Pages 1657-1660

  Li, Bing ^a   Tom, Jeff Y K ^a   Oare, David ^a   Yen, Randy ^a   Fairbrother, Wayne J ^a   Wells, James A ^a   Cunningham, Brian C ^a  

^a GENENTECH INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ATRIAL NATRIURETIC FACTOR; CYSTEINE; DISULFIDE; EPITOPE; PEPTIDE HORMONE;

AMINO ACID SEQUENCE; ARTICLE; HORMONE STRUCTURE; PHAGE DISPLAY; PRIORITY JOURNAL; PROTEIN STRUCTURE;

EID: 0029619255     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.270.5242.1657     Document Type: Article

References (43)

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21. B. C. Cunningham, D. G. Lowe, B. Li, B. D. Bennett, J. A. Wells, EMBO J. 13, 2508 (1994). A phage EUSA was used to quantitate the binding affinity of ANP displaying phage to NPR-A. Alanine mutants of ANP were displayed on the pB1537 phagemid that contained two additional mutations (R3D in ANP and Q29E in the spacer residue immediately after residue 28 of ANP) that increase the efficiency of ANP display without affecting NPR-A-binding affinity (B. Li et al., unpublished data). For Fig. 2 (upper panel) affinities were based on phage ELISA measurements, except those for F8A and I15A, which were determined from radioimmunoassay (RIA) measurements of the respective peptides.
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30. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. Mutations are designated by the wild-type residue, followed by its position and the mutant residue. Multiple mutations are indicated by a series of single mutations separated by hyphens.
31. B. Li and B. C. Cunningham, unpublished data.
32. The decrease (550 times lower) in bioactivity measured for the original 19-aa peptide compared with its 43 times lower receptor affinity may reflect a number of factors, including the susceptibility of this peptide to proteolysis in cell culture or kinetic parameters involved in receptor activation.
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38. Peptides were synthesized on an Applied Biosystems 430A automated synthesizer according to standard N-tert-butoxycarbonyl chemistry protocols, cleaved off the resin with anhydrous HF at 0°C, then cyclized by the potassium ferricyanide method and purified by reversed-phase high-performance liquid chromatography. Masses of each peptide were verified by mass spectrometry. All the peptides except for variant 6 were synthesized with an amidated COOH-terminus.
39. 7 independent mutants and theoretically represented all possible sequence combinations. To minimize wildtype background, the mutagenesis template used for libraries 2 to 5 was frameshifted within the region to be randomized.
40. The molecular models (Fig. 3) are from 1 of 11 energy-minimized structures of an ANP variant (9) whose coordinates are deposited in the Brookhaven Protein Data Bank (accession number 1ANP). This model of wild-type ANP contains two substitutions (L12M-S14R), which revert back mutations that were present in the variant whose structure was solved.
41. B. C. Cunningham, D. G. Lowe, B. Li, B. D. Bennett, J. A. Wells, EMBO J. 13, 2508 (1994); B. D. Bennett et al., J. Biol. Chem. 266, 23060 (1991).
42. B. C. Cunningham, D. G. Lowe, B. Li, B. D. Bennett, J. A. Wells, EMBO J. 13, 2508 (1994); B. D. Bennett et al., J. Biol. Chem. 266, 23060 (1991).
43. We thank D. Lowe for providing the immunoglobulin G-receptor fusion protein and for advice; B. McDowell for discussions; the DNA synthesis group for oligonucleotides; A. Padua for amino acid analysis; and D. Woods, K. Andow, and W. Anstine for help with graphics.