Combinatorial Cassette Mutagenesis as a Probe of the Informational Content of Protein Sequences

Reidhaar-Olson, John F.; Sauer, Robert T.

doi:10.1126/science.3388019

Cited by 331 publications

(175 citation statements)

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“…The amino acid substitutions identified in the 55 high activity variants (group A) are summarized by chemical class in Table 3. Much of the data is consistent with previous studies of mutagenesis of surface residues, 26,27 and with structural and genetic studies of hG-CSF variants specifically. [20][21][22][23][24][25] …”

Section: Sequence Analysis Of Affinity-selected Myelopoietin Variantssupporting

confidence: 80%

“…Sequence analysis of 10 individual phagemids chosen at random from the starting library revealed codon substitutions at each of the mutagenized positions. Though the mutagenesis design should have excluded A and T at the third position of the codons of the mutagenized positionsm, 27 these nucleotides occasionally occurred at that position (occurring twice among the isolates shown in Table 2) and may reflect a minor imperfection in redundant oligonucleotide synthesis.…”

Section: Characterizing and Screening The Myelopoietin Display Librarymentioning

confidence: 99%

“…Oligonucleotide cassettes contained substitutions of the general formula NNK (where N is G, A, T or C and K is either G or C 27 ) at each of the five codons chosen for mutagenesis. Every one of the 20 amino acids usually found in biologically produced proteins can be specified by this approach.…”

Section: Construction Of a Library Of Mutations In The H-gcsf Domain mentioning

confidence: 99%

“…11 Five positions implicated in agonist activity in the hG-CSF receptor agonist module of myelopoietin were mutagenized [20][21][22][23][24][25] using redundant synthetic oligonucleotide cassettes. 26,27 Display experiments efficiently mature affinity of presented ligands for a target, [11][12][13] though maximizing receptor affinity may not maximize cytokine potency. 28 Our screening approach did not restrict us to studying only the high affinity variants [11][12][13] in downstream cell proliferation assays, to allow capture of other desirable variants.…”

Section: Introductionmentioning

confidence: 99%

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Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

Ibdah²,

Valkenburgh

et al. 2001

Leukemia

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Myelopoietins comprise a class of chimeric cytokine receptor agonists consisting of an hIL-3 (human interleukin-3) receptor agonist and an hG-CSF (human granulocyte colony-stimulating factor) receptor agonist linked head-to-tail at their respective carboxy and amino termini. The combination of an early acting cytokine (hIL-3) with a late acting one (hG-CSF) allows efficient hematopoeitic reconstruction following myeloablative insult, and drives differentiation of non-myelocytic lineages (ie thrombocytic lineages) that are inaccessible using hG-CSF alone, in both preclinical models and clinical settings. A myelopoietin species was displayed and mutagenized on filamentous bacteriophage: both component agonists of myelopoietin were presented in biologically functional conformations as each recognized its corresponding receptor. Five amino acid positions in a short region of the hG-CSF receptor agonist module of myelopoietin that had been identified as important for proliferative activity were mutagenized. Display was used because it allows very 'deep' mutagenesis at selected residues: Ͼ10 5 substitution variants were affinity-screened using the hG-CSF receptor and 130 new, active variants of myelopoietin were identified and characterized. None of the selected variants were significantly more active than the parental myelopoietin species in a hG-CSF-dependent cell proliferation assay, though many were as active. Many of these relatively high-activity variants contained parental amino acids at several positions, suggesting the parental sequence may already be optimal at these positions for the assays used, and potentially accounting for the failure to identify enhanced bioactivity variants. Analysis of substitutions of high-activity variants complements and extends previous alanine scanning, and other genetic and biochemical data for hG-CSF variants. Leukemia (2001) 15, 1277-1285.

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Section: Sequence Analysis Of Affinity-selected Myelopoietin Variantssupporting

confidence: 80%

Section: Characterizing and Screening The Myelopoietin Display Librarymentioning

confidence: 99%

Section: Construction Of a Library Of Mutations In The H-gcsf Domain mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

Ibdah²,

Valkenburgh

et al. 2001

Leukemia

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“…Sequence randomization using combinatorial cassette mutagenesis has been used previously to probe the information content of protein sequence of phage repressor (27), and the combinatorial phage library approach has been used for human growth hormone, permitting identification of a recombinant human growth hormone derivative with 400-fold increased receptor binding affinity (18). The same has been achieved with IL-6 (28) and ciliary neurotrophic factor (29), and other phage-cytokine systems such as IL-2 (30) offer similar opportunities.…”

Section: Discussionmentioning

confidence: 99%

Randomization of the Receptor α Chain Recruitment Epitope Reveals a Functional Interleukin-5 with Charge Depletion in the CD Loop

Wu¹,

Tsui

et al. 1999

Journal of Biological Chemistry

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1 is a T cell-derived cytokine that specifically stimulates differentiation, proliferation, and activation of eosinophils (1) and appears to play an important role in the pathogenesis of asthma. IL-5 is a homodimer that folds into a cylindrical molecule containing two four-helix bundles (2), each of which resembles the four-helix bundle seen in the monomeric cytokines IL-3, IL-4, granulocyte-macrophage colony-stimulating factor, and growth hormone.The cell receptor for IL-5 is composed of two subunits, ␣ and ␤ c . The ␣ chain is cytokine-specific and by itself can bind IL-5 with high affinity (3). Extensive site-directed mutagenesis studies have shown that residues clustered near the helix bundle interface, in the CD loop, including Glu 89 and Arg 91 , and at the carboxyl-terminal end of helix D, notably Glu 110 , engage in receptor ␣ chain binding (4 -6). Glu 13 , which is at each of the distal ends of the IL-5 cylinder away from the interface between the two four-helix bundles and is not involved directly in ␣ chain interaction, is a key residue mediating productive interaction of the ␤ c chain of IL-5 receptor (4, 5). It is the ␤ c recruitment into ␣␤ c that leads to triggering of the intracellular signaling cascade.Although site-directed mutagenesis studies have identified important residues for receptor binding, the one-residue-at-atime substitution allowed by this technique limits a full mechanistic understanding of the specific structural and electrostatic features of the IL-5 surface required for receptor-ligand interaction. However, through the use of randomly generated side chain libraries, it should be possible to measure the effect of replacing individual side chains or sets of side chains in local regions on the IL-5 surface and hence determine which combinations allow productive binding. Such random epitope mutagenesis can be achieved with sequence libraries formed by phage display. This technique has been applied successfully to in vitro antibody maturation and protein engineering (7). Typically a foreign gene is fused in frame with phage coat protein pIII encoded by a phagemid vector, and the surface-displayed recombinant foreign protein selected by affinity selection procedures (biopanning). Human growth hormone has been displayed on phage and higher receptor affinity variants selected through random mutagenesis and phage panning (8). Phage display of multichain proteins also has been achieved, for example for antibody Fab fragments (9) and vascular epidermal growth factor (10). Nonetheless, in our hands, phage display of the dimeric wild type IL-5 has proven difficult, perhaps reflecting folding difficulties that previously have led to insoluble inclusion bodies in intracellular Escherichia coli expression (11).We have sought to overcome limitations in phage display of IL-5 by using a single chain form of human IL-5 (scIL-5), in which two IL-5 molecules are tandemly linked by a Gly-Gly dipeptide linker (12). Single chain IL-5 and wtIL-5 have virtually identical biological activities and bindin...

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Constructing amino acid residue substitution classes maximally indicative of local protein structure

Thompson

Goldstein

1996

Proteins

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Using an information theoretic formalism, we optimize classes of amino acid substitution to be maximally indicative of local protein structure. Our statistically‐derived classes are loosely identifiable with the heuristic constructions found in previously published work. However, while these other methods provide a more rigid idealization of physicochemically constrained residue substitution, our classes provide substantially more structural information with many fewer parameters. Moreover, these substitution classes are consistent with the paradigmatic view of the sequence‐to‐structure relationship in globular proteins which holds that the three‐dimensional architecture is predominantly determined by the arrangement of hydrophobic and polar side chains with weak constraints on the actual amino acid identities. More specific constraints are imposed on the placement of prolines, glycines, and the charged residues. These substitution classes have been used in highly accurate predictions of residue solvent accessibility. They could also be used in the identification of homologous proteins, the construction and refinement of multiple sequence alignments, and as a means of condensing and codifying the information in multiple sequence alignments for secondary structure prediction and tertiary fold recognition. © 1996 Wiley‐Liss, Inc.

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Combinatorial Cassette Mutagenesis as a Probe of the Informational Content of Protein Sequences

Cited by 331 publications

References 29 publications

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

Phage display mutagenesis of the chimeric dual cytokine receptor agonist myelopoietin

Randomization of the Receptor α Chain Recruitment Epitope Reveals a Functional Interleukin-5 with Charge Depletion in the CD Loop

Constructing amino acid residue substitution classes maximally indicative of local protein structure

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