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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/102—Mutagenizing nucleic acids
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1044—Preparation or screening of libraries displayed on scaffold proteins
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1089—Design, preparation, screening or analysis of libraries using computer algorithms
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• • C—CHEMISTRY; METALLURGY
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  • C12Y—ENZYMES
  • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
  • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
  • C12Y305/02006—Beta-lactamase (3.5.2.6)

Definitions

• the invention relates to the use of protein design automation (PDA) to generate computationally prescreened secondary libraries of proteins, and to methods and compositions utilizing the libraries
• Directed molecular evolution can be used to create proteins and enzymes with novel functions and properties Starting with a known natural protein, several rounds of mutagenesis, functional screening, and propagation of successful sequences are performed The advantage of this process is that it can be used to rapidly evolve any protein without knowledge of its structure Several different mutagenesis strategies exist, including point mutagenesis by error-prone PCR, cassette mutagenesis, and DNA shuffling These techniques have had many successes, however, they are all handicapped by their inability to produce more than a tiny fraction of the potential changes For example, there are 20 500 possible ammo acid changes for an average protein approximately 500 ammo acids long Clearly, the mutagenesis and functional screening of so many mutants is impossible, directed evolution provides a very sparse sampling of the possible sequences and hence examines only a small portion of possible improved proteins, typically point mutants or recombinations of existing sequences By sampling randomly from the vast number of possible sequences, directed evolution is unbiased and broadly applicable, but inherently inefficient because it ignores all structural and bio
• sequence profiling Bowie and Eisenberg, Science 253(5016) 164-70, (1991)
• rotamer library selections Disahiyat and Mayo, Protein Set 5(5) 895-903 (1996), Dahiyat and Mayo, Science 278(5335) 82-7 (1997), Desjarlais and Handel, Protein Science 4 2006-2018 (1995), Harbury et al, PNAS USA 92(18) 8408-8412 (1995), Kono et al , Proteins Structure, Function and Genetics 19 244-255 (1994), Hellmga and Richards, PNAS USA 91 5803-5807 (1994)), and residue pair potentials (Jones, Protein Science 3 567-574, (1994))
• the present invention provides methods for generating a secondary library of scaffold protein variants comprising providing a primary library comprising a rank-ordered list of scaffold protein primary variant sequences A list of primary variant positions in the primary library is then generated, and a plurality of the primary variant positions is then combined to generate a secondary library of secondary sequences
• the invention provides methods for generating a secondary library of scaffold protein variants comprising providing a primary library comprising a rank-ordered list of scaffold protein primary variant sequences, and generating a probability distribution of ammo acid residues in a plurality of variant positions The plurality of the ammo acid residues is combined to generate a secondary library of secondary sequences
• sequences may then be optionally synthesized and tested, in a variety of ways, including multiplexing PCR with pooled oligonucleotides, error prone PCR, gene shuffling, etc
• the invention provides compositions comprising a plurality of secondary variant proteins or nucleic acids encoding the proteins, wherein the plurality comprises all or a subset of the secondary library
• the invention further provides cells comprising the library, particularly mammalian cells
• the invention provides methods for generating a secondary library of scaffold protein variants comprising providing a first library rank-ordered list of scaffold protein primary variants, generating a probability distribution of ammo acid residues in a plurality of variant positions, and synthesizing a plurality of scaffold protein secondary variants comprising a plurality of the ammo acid residues to form a secondary library At least one of the secondary variants is different from the primary variants
• the present invention provides methods executed by a computer under the control of a program, the computer including a memory for storing the program
• the method comprising the steps of receiving a protein backbone structure with variable residue positions, establishing a group of potential rotamers for each of the variable residue positions, and analyzing the interaction of each of the rotamers with all or part of the remainder of the protein backbone structure to generate a set of optimized protein sequences
• the methods further comprise classifying each variable residue position as either a core, surface or boundary residue
• the analyzing step may include a Dead-End Elimination (DEE) computation
• the analyzing step includes the use of at least one scoring function selected from the group consisting of a Van der Waals potential scoring function, a hydrogen bond potential scoring function, an atomic solvation scoring function, a secondary structure propensity scoring function and an electrostatic scoring function
• the methods further comprise altering the protein backbone prior to the analysis, comprising altering at least one supersecondary structure parameter value
• the methods may further comprise generating a rank
• Figure 1 depicts the synthesis of a full-length gene and all possible mutations by PCR Overlapping oligonucleotides corresponding to the full-length gene (black bar, Step 1 ) are synthesized, heated and annealed Addition of Pfu DNA polymerase to the annealed oligonucleotides results in the 5' -3' synthesis of DNA (Step 2) to produce longer DNA fragments (Step 3) Repeated cycles of heating, annealing (Step 4) results in the production of longer DNA, including some full-length molecules These can be selected by a second round of PCR using primers (arrowed) corresponding to the end of the full-length gene (Step 5)
• Figure 2 depicts the reduction of the dimensionality of sequence space by PDA screening From left to right, 1 without PDA, 2 without PDA not counting Cysteine, Proline, Glycine, 3 with PDA using the 1% criterion, modeling free enzyme, 4 with PDA using the 1% criterion, modeling enzyme-substrate complex, 5 with PDA using the 5% criterion modeling free enzyme, 6 with PDA using the 5% criterion modeling enzyme-substrate complex
• Figure 3 depicts the active site of B circulans xylanase Those positions included in the PDA design are shown by their side chain representation In red are wild type residues (their conformation was allowed to change, but not their am o acid identity) In green are positions whose conformation and identity were allowed to change (to any ammo acid except proline, cysteine and glycine)
• FIG. 4 depicts cefotaxime resistance of E coll expressing wild type (WT) and PDA Screened ⁇ -lactamase, results shown for increasing concentrations of cefotaxime
• the present invention is directed to methods of using computational screening of protein sequence libraries (that can comprise up to 10 80 or more members) to select smaller secondary libraries of protein sequences (that can comprise up to 10 13 members), that can then be actually synthesized and experimentally tested in the desired assay, for improved function and properties
• the invention has two broad uses, first, the invention can be used to prescreen libraries based on known scaffold proteins That is, computational screening for stability (or other properties) may be done on either the entire protein or some subset of residues, as desired and described below
• computational screening for stability or other properties
• the percentage of useful variants in a given variant set size can increase, and the required experimental outlay is decreased
• the present invention finds use in the screening of random peptide libraries
• signaling pathways in cells often begin with an effector stimulus that leads to a phenotypically describable change in cellular physiology
• intracellular signaling pathways play in disease pathogenesis, in most cases, little is understood about a signaling pathway other than the initial stimulus and the ultimate cellular response
• sequences in these experimental libraries can be randomized at specific sites only, or throughout the sequence
• the number of sequences that can be searched in these libraries grows expontentially with the number of positions that are randomized
• 10 12 - 10 15 sequences can be contained in a library because of the physical constraints of laboratories (the size of the instruments, the cost of producing large numbers of biopolymers, etc )
• Other practical considerations can often limit the size of the libraries to 10 6 or fewer These limits are reached for only 10 ammo acid positions
• novel methods to create secondary libraries derived from very large computational mutant libraries allow the rapid testing of large numbers of computationally designed sequences
• the libraries may be biased in any number of ways, allowing the generation of secondary libraries that vary in their focus, for example, domains, subsets of residues, active or binding sites, surface residues, etc , may all be varied or kept constant as desired
• protein herein is meant at least two am o acids linked together by a peptide bond
• protein includes proteins, oligopeptides and peptides
• the peptidyl group may comprise naturally occurring ammo acids and peptide bonds, or synthetic peptidomimetic structures, i e "analogs", such as peptoids (see Simon ef al , PNAS USA 89(20) 9367 (19-92))
• the ammo acids may either be naturally occu ⁇ ng or non-naturally occu ⁇ ng, as will be appreciated by those in the art, any structure for which a set of rotamers is known or can be generated can be used as an ammo acid
• the side chains may be in either the (R) or the (S) configuration
• the ammo acids are in the (S) or L-configuration
• the scaffold protein may be any protein for which a three dimensional structure is known or can be generated, that is, for which there are three dimensional coordinates for each atom of the protein Generally this can be determined using X-ray crystallographic techniques, NMR techniques, de novo modelling, homology modelling, etc In general, if X-ray structures are used, structures at 2 ⁇ resolution or better are preferred, but not required
• the scaffold proteins may be from any organism, including prokaryotes and eukaryotes, with enzymes from bacteria, fungi, extremeophiles such as the archebacte ⁇ a, insects, fish, animals (particularly mammals and particularly human) and birds all possible
• scaffold protein herein is meant a protein for which a secondary library of variants is desired
• any number of scaffold proteins find use in the present invention Specifically included within the definition of “protein” are fragments and domains of known proteins, including functional domains such as enzymatic domains, binding domains, etc , and smaller fragments, such as turns, loops, etc That is, portions of proteins may be used as well
• “protein” as used herein includes proteins, oligopeptides and peptides
• protein variants i e non-naturally occu ⁇ ng protein analog structures, may be used Suitable proteins include, but are not limited to, industrial and pharmaceutical proteins, including ligands, cell surface receptors, antigens, antibodies, cytokmes, hormones, transcription factors, signalling modules, cytoskeletal proteins and enzymes Suitable classes of enzymes include, but are not limited to, hydrolases such as proteases, carbohydrases, lipases, isomerase
• preferred scaffold proteins include, but are not limited to, those with known structures (including variants) including cytokmes (IL-1 ra (+receptor complex), IL-1 (receptor alone), IL-1a, IL-1 b
• IL-2 (including variants and or receptor complex), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IFN- ⁇ , INF- ⁇ , IFN- ⁇ -2a, IFN- ⁇ -2B, TNF- ⁇ , CD40 ligand (chk), Human Obesity Protein Leptm, Granulocyte Colony- Stimulating Factor, Bone Morphogenetic Prote ⁇ n-7, Ciliary Neurotrophic Factor, Granulocyte- Macrophage Colony-Stimulating Factor, Monocyte Chemoattractant Protein 1 , Macrophage Migration Inhibitory Factor, Human Glycosylation-lnhibitmg Factor, Human Rantes, Human Macrophage
• Inflammatory Protein 1 Beta human growth hormone, Leukemia Inhibitory Factor, Human Melanoma Growth Stimulatory Activity, neutrophil activating pept ⁇ de-2, Cc-Chemokme Mcp-3, Platelet Factor M2, Neutrophil Activating Peptide 2, Eotaxin, Stromal Cell-Derived Factor-1 , Insulin, Insulin-like Growth Factor I, Insulin-like Growth Factor II, Transforming Growth Factor B1 , Transforming Growth Factor B2, Transforming Growth Factor B3, Transforming Growth Factor A, Vascular Endothelial growth factor (VEGF), acidic Fibroblast growth factor, basic Fibroblast growth factor, Endothelial growth factor, Nerve growth factor, Brain Derived Neurotrophic Factor, Ciliary Neurotrophic Factor, Platelet Derived Growth Factor, Human Hepatocyte Growth Factor, Glial Cell-Derived Neurotrophic Factor, (as well as the 55 cytokmes in PDB 1/12/99)), Erythro
• a primary library is generated using computational processing
• the goal of the computational processing is to determine a. set of optimized protein sequences
• a global optimized sequence is the one sequence that best fits the equations (for example, when PDA is used, the global optimzed sequence is the sequence that best fits Equation 1 , b-elow), i e the sequence that has the lowest energy of any possible sequence
• a "primary library” as used herein is a collection of optimized sequences, generally in the form of a rank-ordered list
• all possible sequences of a protein may be ranked, however, currently 10 13 sequences is a practical limit
• some subset of all possible sequences is used as the primary library, generally, the top 10 3 to 10 13 sequences are chosen as the primary library
• the cutoff for inclusion in the rank ordered list of the primary library can be done in a variety of ways
• the cutoff may be just an arbitrary exclusion point the top 10 5 sequences may comprise the primary library
• all sequences scoring within a certain limit of the global optimum can be used, for example, all sequences with 10 kcal/mol of the global optimum could be used as the primary library
• This method has the advantage of using a direct measure of fidelity to a three dimensional structure to determine inclusion This approach can be used to insure that library mutations are not limited to positions that have the lowest energy gap between different mutations Alternatively, the cutoff may be enforce
• the primary libraries can be generated in a variety of ways In essence, any methods that can result in the relative ranking of the possible sequences of a protein based on measurable stability parameters can be used As will be appreciated by those in the art, any of the methods described herein or known in the art may be used alone, or in combination with other methods
• the scaffold protein is an enzyme and highly accurate electrostatic models can be used for enzyme active site residue scoring to improve enzyme active site libraries (see Warshel, computer Modeling of Chemical Reactions in Enzymes and Solutions. Wiley & Sons, New York, (1991), hereby expressly incorporated by reference) These accurate models can assess the relative energies of sequences with high precision, but are computationally intensive
• molecular dynamics calculations can be used to computationally screen sequences by individually calculating mutant sequence scores and compiling a rank ordered list
• residue pair potentials can be used to score sequences (Miyazawa et al Macromolecules 18(3) 534-552 (1985), expressly incorporated by reference) during computational screening
• sequence profile scores (Bowie et al , Science 253(5016) 164-70 (1991), incorporated by reference) and/or potentials of mean force (Hend ch et al , J Mol Biol 216(1) 167- 180 (1990), also incorporated by reference) can also be calculated to score sequences
• scoring functions can be used to screen for sequences that would create metal or co- factor binding sites in the protein (Hellmga, Fold Des 3(1) R1-8 (1998), hereby expressly incorporated by reference)
• scoring functions can be used to screen for sequences that would create disulfide bonds in the protein
• sequence and/or structural alignment programs can be used to generate primary libraries For example, structural alignment of structurally related proteins can be done to generate sequence alignments (Orengo et al , Structure 5(8) 1093-108 (1997), Holm et al , Nucleic-Acid Res 26(1) 316-9 (1998), both of which are incorporated by reference) These sequence alignments can then be examined to determine the observed sequence variations
• sequence homology based alignment methods can be used to create sequence alignments of proteins related to the target structure (Altschul et al , J Mol Biol 215(3) 403 (1990), incorporated by reference) These sequence alignments are then examined to determine the observed sequence variations These sequence variations are tabulated to define a primary library
• sequence variations can be tabulated and a secondary library defined from them as defined below Alternatively, the allowed sequence variations can be used to define the ammo acids considered at each position during the computational screening Another variation is to bias the score for ammo acids that occur in the sequence alignment, thereby increasing the likelihood that they are found during computational screening but still allowing consideration of other ammo acids This bias would result in a focused primary library but would not eliminate from consideration ammo acids not found in the alignment
• the computational method used to generate the primary library is Protein Design Automation (PDA), as is described in U S S N s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926 and PCT US98/07254, all of which are expressly incorporated herein by reference
• PDA Protein Design Automation
• a known protein structure is used as the starting point
• the residues to be optimized are then identified, which may be the entire sequence or subset(s) thereof
• Each variable residue position is then preferably classified as a core residue, a surface residue, or a boundary residue, each classification defines a subset of possible ammo acid residues for the position (for example, core residues generally will be selected from the set of hydrophobic residues, surface residues generally will be selected from the hydrophilic residues, and boundary residues may be either)
• Each ammo acid can be represented by a discrete set of all allowed conformers of each side chain, called rotamers
• all possible sequences of rotamers must be screened, where each backbone position can be occupied either by each ammo acid in all its possible rotame ⁇ c states, or a subset of ammo acids, and thus a subset of rotamers
• a Monte Carlo search may be done to generate a rank- ordered list of sequences in the neighborhood of the DEE solution
• Starting at the DEE solution random positions are changed to other rotamers, and the new sequence energy is calculated If the new sequence meets the criteria for acceptance, it is used as a starting point for another jump After a predetermined number of jumps, a rank-ordered list of sequences is generated
• the protein backbone (comprising (for a naturally occu ⁇ ng protein) the nitrogen, the carbonyl carbon, the ⁇ -carbon, and the carbonyl oxygen, along with the direction of the vector from the ⁇ -carbon to the ⁇ -carbon) may be altered prior to the computational analysis, by varying a set of parameters called supersecondary structure parameters
• the protein backbone structure contains at least one variable residue position
• residues, or ammo acids, of proteins are generally sequentially numbered starting with the N- term us of the protein
• a protein having a methionine at it's N-terminus is said to have a methionine at residue or ammo acid position 1 , with the next residues as 2, 3, 4, etc
• the wild type (i e naturally occu ⁇ ng) protein may have one of at least 20 ammo acids, in any number of rotamers
• variable residue position herein is meant an ammo acid position of the protein to be designed that is not fixed in the design method as a specific residue or rotamer, generally the wild-type residue or rotamer
• all of the residue positions of the protein are variable That is, every ammo acid side chain may be altered in the methods of the present invention This is particularly desirable for smaller proteins, although the present methods allow the design of larger proteins as well While there is no theoretical limit to the length of
• residue positions of the protein are variable, and the remainder are "fixed", that is, they are identified in the three dimensional structure as being in a set conformation
• a fixed position is left in its original conformation (which may or may not correlate to a specific rotamer of the rotamer library being used)
• residues may be fixed as a non-wild type residue, for example, when known site-directed mutagenesis techniques have shown that a particular residue is desirable (for example, to eliminate a proteolytic site or alter the substrate specificity of an enzyme), the residue may be fixed as a particular ammo acid
• the methods of the present invention may be used to evaluate mutations de novo, as is discussed below
• a fixed position may be "floated", the ammo acid at that position is fixed, but different rotamers of that ammo acid are tested
• the variable residues may be at least one, or anywhere from 0 1 % to 99 9% of the total
• residues which can be fixed include, but are not limited to, structurally or biologically functional residues
• residues which are known to be important for biological activity such as the residues which form the active site of an enzyme, the substrate binding site of an enzyme, the binding site for a binding partner (ligand/receptor, antigen/antibody, etc ), phosphorylation or glycosylation sites which are crucial to biological function, or structurally important residues, such as disulfide bridges, metal binding sites, critical hydrogen bonding residues, residues critical for backbone conformation such as proline or glycine, residues critical for packing interactions, etc may all be fixed in a conformation or as a single rotamer, or "floated"
• variable residues which may be chosen as variable residues may be those that confer undesirable biological attributes, such as susceptibility to proteolytic degradation, dime ⁇ zation or aggregation sites, glycosylation sites which may lead to immune responses, unwanted binding activity, unwanted allostery, undesirable enzyme activity but with a preservation of binding, etc
• each variable position is classified as either a core, surface or boundary residue position, although in some cases, as explained below, the variable position may be set to glycine to minimize backbone strain
• Any combination of core, surface and boundary positions can be utilized core, surface and boundary residues, core and surface residues, core and boundary residues, and surface and boundary residues, as well as core residues alone, surface residues alone, or boundary residues alone
• the classification of residue positions as core, surface or boundary may be done in several ways, as will be appreciated by those in the art In a preferred embodiment, the classification is done via a visual scan of the original protein backbone structure, including the side chains, and assigning a classification based on a subjective evaluation of one skilled in the art of protein modelling
• a preferred embodiment utilizes an assessment of the orientation of the C ⁇ -C ⁇ vectors relative to a solvent accessible surface computed using only the template C ⁇ atoms, as outlined in U S S N s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926 and PCT US98/07254
• a core residue will generally be selected from the group of hydrophobic residues consisting of alanme, valme, isoleucme, leucme, phenylalanme, tyrosine, tryptophan, and methionine (in some embodiments, when the ⁇ scaling factor of the van der Waals scoring function, described below, is low, methionine is removed from the set), and the rotamer set for each core position potentially includes rotamers for these eight ammo acid side chains (all the rotamers if a backbone independent library is used, and subset
• proline, cysteine and glycine are not included in the list of possible ammo acid side chains, and thus the rotamers for these side chains are not used
• the variable residue position has a ⁇ angle (that is, the dihedral angle defined by 1) the carbonyl carbon of the preceding ammo acid, 2) the nitrogen atom of the current residue, 3) the ⁇ - carbon of the current residue, and 4) the carbonyl carbon of the current residue) greater than 0°
• the position is set to glycine to minimize backbone strain
• processing proceeds as outlined in U S S N 09/127,926 and PCT US98/07254
• This processing step entails analyzing interactions of the rotamers with each other and with the protein backbone to generate optimized protein sequences Simplistically, the processing initially comprises the use of a number of scoring functions to calculate energies of interactions of the rotamers, either to the backbone itself or other rotamers
• Preferred PDA scoring functions include, but are not limited to, a Van der Waals potential scoring function, a hydrogen bond potential scoring function, an atomic solvation scoring function, a secondary structure propensity scoring function and an electrostatic scoring function
• at least one scoring function is used to score each position, although the scoring functions may differ depending on the position classification or other considerations, like favorable interaction with an ⁇ -he x dipole
• the total energy which is used in the calculations is the sum of the energy of each scoring function used at a particular position, as is generally shown in
• Equation 1 the total energy is the sum of the energy of the van der Waals potential (E vdw ), the energy of atomic solvation (E as ), the energy of hydrogen bonding (E h bond , ng ), the energy of secondary structure (E ss ) and the energy of electrostatic interaction (E etec )
• E vdw van der Waals potential
• E as the energy of atomic solvation
• E h bond the energy of hydrogen bonding
• E ss the energy of secondary structure
• E etec the energy of electrostatic interaction
• the preferred first step in the computational analysis comprises the determination of the interaction of each possible rotamer with all or part of the remainder of the protein That is, the energy of interaction, as measured by one or more of the scoring functions, of each possible rotamer at each variable residue position with either the backbone or other rotamers, is calculated
• the interaction of each rotamer with the entire remainder of the protein, i e both the entire template and all other rotamers is done
• the first step of the computational processing is done by calculating two sets of interactions for each rotamer at every position the interaction of the rotamer side chain with the template or backbone (the “singles” energy), and the interaction of the rotamer side chain with all other possible rotamers at every other position (the “doubles” energy), whether that position is varied or floated
• the backbone in this case includes both the atoms of the protein structure backbone, as well as the atoms of any fixed residues, wherein the fixed residues are defined as a particular conformation of an ammo acid
• DEE Dead End Elimination
• each optimized protein sequence preferably comprises at least about 5-10% variant ammo acids from the starting or wild-type sequence, with at least about
• the cutoff for the primary library is then enforced, resulting in a set of primary sequences forming the primary library
• this may be done in a variety of ways, including an arbitrary cutoff, an energy limitation, or when a certain number of residue positions have been varied
• the size of the primary library will vary with the size of the protein, the number of residues that are changing, the computational methods used, the cutoff applied and the discretion of the user
• primary libraries that range from about 50 to about 10 13 are preferred, with from about 1000 to about 10 7 being particularly preferred, and from about 1000 to about 100,000 being especially preferred
• the primary library comprises the globally optimal sequence in its optimal conformation, i e the optimum rotamer at each variable position That is, computational processing is run until the simulation program converges on a single sequence which is the global optimum
• the primary library comprises at least two optimized protein sequences
• the computational processing step may eliminate a number of disfavored combinations but be stopped prior to convergence, providing a library of sequences of which the global optimum is one
• further computational analysis for example using a different method, may be run on the library, to further eliminate sequences or rank them differently Alternatively, as is more fully described in U S S N s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926 and PCT US98/07254, the global optimum may be reached, and then further computational processing may occur, which generates additional optimized sequences in the neighborhood of the global optimum
• primary library sequences that did not make the cutoff are included in the primary library This may be desirable in some situations to evaluate the primary library generation method, to serve as controls or comparisons, or to sample additional sequence space
• the wild-type sequence is included
• the present invention provides primary libraries comprising a rank ordered list of sequences
• all or a portion of the primary library serves as the secondary library That is, a cutoff is applied to the primary sequences and these sequences serve as the secondary library, without further manipulation or recombination
• the library members can be made as outlined below, e g by direct synthesis or by constructing the nucleic acids encoding the library members, expressing them in a suitable host, optionally followed by screening
• the primary library of the scaffold protein is used to generate a secondary library
• the secondary library can be either a subset of the primary Imrary, or contain new library members, i e sequences that are not found in the primary library That is, in general, the variant positions and/or ammo acid residues in the variant positions can be recombmed in any number of ways to form a new library that exploits the sequence variations found in the primary library That is, having identified "hot spots" or important variant positions and/or residues, these positions can be recombmed in novel ways to generate novel sequences to form a secondary library
• the secondary library comprises at least one member sequence that is not found in the primary library, and preferably a plurality of such sequences
• the secondary library is generated by tabulating the ammo acid positions that vary from a reference sequence
• the reference sequence can be arbitrarily selected, or preferably is chosen either as the wild-type sequence or the global optimum sequence, with the latter being preferred That is, each ammo acid position that varies in the primary library is tabulated
• the variable positions of the secondary library will comprise either just these original variable positions or some subset of these original variable positions That is, assuming a protein of 100 ammo acids, the original computational screen can allow all 100 positions to be varied However, due to the cutoff in the primary library, only 25 positions may vary Alternatively, assuming the same 100 ammo acid protein, the original computational screen could have varied only 25 positions, keeping the other 75 fixed, this could result in only 12 of the 25 being varied in the cutoff primary library
• These primary library positions can then be recombmed to form a secondary library, wherein all possible combinations of these variable positions form the secondary library It should be noted that the non-variable positions are set
• the formation of the secondary library using this method may be done in two general ways, either all variable positions are allowed to be any ammo acid, or subsets of ammo acids are allowed for each position In a preferred embodiment, all ammo acid residues are allowed at each variable position identified in the primary library That is, once the variable positions are identified, a secondary library comprising every combination of every ammo acid at each variable position is made
• subsets of ammo acids are chosen The subset at any position may be either chosen by the user, or may be a collection of the ammo acid residues generated in the primary screen That is, assuming core residue 25 is variable and the primary screen gives 5 different possible ammo acids for this position, the user may chose the set of good core residues outlined above (e g hydrophobic residues), or the user may build the set by chosing the 5 different ammo acids generated in the primary screen Alternatively, combinations of these techniques may be used, wherein the set of identified residues is manually expanded For example, in some embodiments, fewer than the number of ammo acid residues is chosen, for example, only three of the five may be chosen Alternatively, the set is manually expanded, for example, if the computation picks two different hydrophobic residues, additional choices may be added
• the secondary library can be generated by randomizing the ammo acids at the positions that have high numbers of mutations, while keeping constant the positions that do not have mutations above a certain frequency For example, if the position has less than 20% and more preferably 10% mutations, it may be kept constant as the reference sequence position
• a probability distribution table is generated
• the frequency of each ammo acid residue at each variable position is identified
• Frequencies can be thresholded, wherein any variant frequency lower than a cutoff is set to zero
• This cutoff is preferably 1 %, 2%, 5%, 10% or 20%, with 10% being particularly preferred
• these frequencies are then built into the secondary library That is, as above, these variable positions are collected and all possible combinations are generated, but the ammo acid residues that "fill" the secondary library are utilized on a frequency basis
• a variable position that has 5 possible residues will have 20% of the proteins comprising that variable position with the first possible residue, 20% with the second, etc
• a variable position that has 5 possible residues with frequencies of 10%, 15%, 25%, 30% and 20%, respectively, will have 10% of the proteins comprising that variable position with the first possible residue, 15% of the proteins with the second residue, 25% with the third, etc
• a secondary library created by recombining variable positions and/or residues at the variable position may not be in a rank-ordered list. In some embodiments, the entire list may just be made and tested. Alternatively, in a preferred embodiment, the secondary library is also in the form of a rank ordered list. This may be done for several reasons, including the size of the secondary library is still too big to generate experimentally, or for predictive purposes. This may be done in several ways. In one embodiment, the secondary library is ranked using the scoring functions of PDA to rank the library members. Alternatively, statistical methods could be used. For example, the secondary library may be ranked by frequency score; that is, proteins containing the most of high frequency residues could be ranked higher, etc. This may be done by adding or multiplying the frequency at each variable position to generate a numerical score. Similarly, the secondary library different positions could be weighted and then the proteins scored; for example, those containing certain residues could be artbitrarily ranked.
• secondary libraries can be generated in two general ways. The first is computationally, as above, wherein the primary library is further computationally manipulated, for example by recombining the possible variant positions and/or amino acid residues at each variant position. It may be ranked, as outlined above.
• This computationally-derived secondary library can then be experimentally generated by synthesizing the library members or nucleic acids encoding them, as is more fully outlined below.
• the secondary library is made experimentally; that is, nucleic acid recombination techniques are used to experimentally generate the combinations. This can be done in a variety of ways, as outlined below.
• the different protein members of the secondary library may be chemically synthesized. This is particularly useful when the designed proteins are short, preferably less than 150 amino acids in length, with less than 100 amino acids being preferred, and less than 50 amino acids being particularly preferred, although as is known in the art, longer proteins can be made chemically or enzymatically. See for example Wilken et al, Curr. Opin. Biotechnol. 9:412-26 (1998), hereby expressly incorporated by reference.
• the secondary library sequences are used to create nucleic acids such as DNA which encode the member sequences and which can then be cloned into host cells, expressed and assayed, if desired
• nucleic acids, and particularly DNA can be made which encodes each member protein sequence This is done using well known procedures
• codons, suitable expression vectors and suitable host cells will vary depending on a number of factors, and can be easily optimized as needed
• oligonucleotides are synthesized which correspond to the full length gene
• these oligonucleotides may represent all of the different ammo acids at each variant position or subsets
• these oligonucleotides are pooled in equal proportions and multiple PCR reactions are performed to create full length sequences containing the combinations of mutations defined by the secondary library
• the different oligonucleotides are added in relative amounts corresponding to the probability distribution table
• the multiple PCR reactions thus result in full length sequences with the desired combinations of mutaions in the desired proportions
• each overlapping o gonucleotide comprises only one position to be varied, in alternate embodiments, the variant positions are too close together to allow this and multiple variants per ohgonucleotide are used to allow complete recombination of all the possibilities That is, each o go can contain the codon for a single position being mutated, or for more than one position being mutated The multiple positions being mutated must be close in sequence to prevent the o go length from being impractical For multiple mutating positions on an ohgonucleotide, particular combinations of mutations can be included or excluded in the library by including or excluding the ohgonucleotide encoding that combination The total number of oligonucleotides required increases when multiple mutable positions are encoded by a single ohgonucleotide The annealed regions are the ones that remain constant, i e have the sequence of the reference sequence
• Oligonucleotides with insertions or deletions of codons can be used to create a library expressing different length proteins
• computational sequence screening for insertions or deletions can result in secondary libraries defining different length proteins, which can be expressed by a library of pooled ohgonucleotide of different lengths
• error-prone PCR is done to generate the secondary library See U S
• Patent Nos 5,605,793, 5,811 ,238, and 5,830,721 all of which are hereby incorporated by reference
• This can be done on the optimal sequence or on top members of the library
• the gene for the optimal sequence found in the computational screen of the primary library can be synthesized Error prone PCR is then performed on the optimal sequence gene in the presence of oligonucleotides that code for the mutations at the variant positions of the secondary library (bias oligonucleotides)
• bias oligonucleotides The addition of the oligonucleotides will create a bias favoring the incorporation of the mutations in the secondary library
• only oligonucleotides for certain mutations may be used to bias the library
• gene shuffling with error prone PCR can be performed on the gene for the optimal sequence, in the presence of bias oligonucleotides, to create a DNA sequence library that reflects the proportion of the mutations found in the secondary library
• bias oligonucleotides can be done in a variety of ways, they can chosen on the basis of their frequency, i e oligonucleotides encoding high mutational frequency positions can be used, alternatively, oligonucleotides containing the most variable positions can be used, such that the diversity is increased, if the secondary library is ranked, some number of top scoring positions can be used to generate bias oligonucleotides, random positions may be chosen, a few top scoring and a few low scoring ones may be chosen, etc What is important is to generate new sequences based on preferred variable positions and sequences
• a secondary library may be computationally remanipulated to form an additional secondary library
• any of the secondary library sequences may be chosen for a second round of PDA, by freezing or fixing some or all of the changed positions in the first secondary library
• the stringency of the probability table may be altered, either by increasing or decreasing the cutoff for inclusion
• the secondary library may be recombmed experimentally after the first round, for example, the best gene/genes from the first screen may be taken and gene assembly redone (using techniques outlined below, multiple PCR, error prone PCR, shuffling, etc )
• the fragments from one or more good gene(s) to change probabilities at some positions This biases the search to an area of sequence space found in the first round of computational and experimental screening
• any number of protein attributes may be altered in these methods, including, but not limited to, enzyme activity, stability, solubility, aggregation, binding affinity, binding specificity, substrate specificity, structural integrity, immunogenicity, toxicity, generate peptide and peptidomimmetic libraries, create new antibody CDR's, generate new DNA, RNA bindings, etc
• therapeutic proteins utilized in these methods will preferentially have residues in the hydrophobic cores screened, to prevent changes in the molecular surface of the protein that might induce immunoge ⁇ ic responses
• Therapeutic proteins cna also be designed in the region surrounding their binding sites to their receptors Such a region can be defined, for example, by including in the design all residues within a certain distance, for example 4 5 A of the binding site residues This range can vary from 4 to 6 A This designe will serve to improve enzyme activity and specificity
• the methods of the invention are used not on known scaffold proteins, but on random peptides, to search a virtual library for those sequences likely to adapt a stable conformation
• the sequences in these experimental libraries can be randomized at specific sites only, or throughout the sequence The number of sequences that can be searched in these libraries grows expontentially with the number of positions that are randomized
• the automated protein design generates a list of sequence candidates that are favored to meet design criteria, it also shows which positions in the sequence are readily changed and which positions are unlikely to change without disrupting protein stability and function
• An experimental random library can be generated that is only randomized at the readily changeable, non-disruptive sequence positions
• the diversity of ammo acids at these positions can be limited to those that the automated design shows are compatible with these positions
• positions 9, 10 and 12 are most likely to have changes that do not disrupt the function of the protein, suggesting that a random experimental library that randomizes positions 9, 10 and 12 will have a higher fraction of desirable sequences Also, the virtual library suggests that position 10 is most compatible with lie or Phe residues, further limiting the size of the library and allowing a more complete screening of good sequences
• the automated design method uses physical chemical criteria to screen sequences, resulting in sequences that are likely to be stable, structured, and that preserve function, if needed.
• Different design criteria can be used to produce candidate sets that are biased for properties such as charged, solubility, or active site characteristics (polarity, size), or are biased to have certain amino acids at certain positions. That is,
• the candidate bioactive agents and candidate nucleic acids are randomized, either fully randomized or they are biased in their randomization, e.g. in nucleotide/residue frequency generally or per position.
• randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Thus, any amino acid residue may be incorporated at any position.
• the synthetic process can be designed to generate randomized peptides and/or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized candidate nucleic
• the library is fully randomized, with no sequence preferences or constants at any position.
• the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
• the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
• the bias is towards peptides or nucleic acids that interact with known classes of molecules.
• a short region from the HIV-1 envelope cytoplasmic domain has been previously shown to block the action of cellular calmodulin.
• Regions of the Fas cytoplasmic domain which shows homology to the mastoparan toxin from Wasps, can be limited to a short peptide region with death- inducing apoptotic or G protein inducing functions.
• Magainin a natural peptide derived from Xenopus, can have potent anti-tumour and anti-microbial activity.
• Short peptide fragments of a protein kinase C isozyme ( ⁇ PKC) have been shown to block nuclear translocation of ⁇ PKC in Xenopus oocytes following stimulation.
• ⁇ PKC protein kinase C isozyme
• SH-3 target peptides have been used as psuedosubstrates for specific binding to SH-3 proteins. This is of course a short list of available peptides with biological activity, as the literature is dense in this area. Thus, there is much precedent for the potential of small peptides to have activity on intracellular signaling cascades.
• agonists and antagonists of any number of molecules may be used as the basis of biased randomization of candidate bioactive agents as well.
• a prescreened random peptide libraries may be described as follows. Any structure, whether a known structure, for example a portion of a known protein, a known peptide, etc., or a synthetic structure, can be used as the backbone for PDA. For example, structures from X- ray crystallographic techniques, NMR techniques, de novo modelling, homology modelling, etc. may all be used to pick a backbone for which sequences are desired. Similarly, a number of molecules or protein domains are suitable as starting points for the generation of biased randomized candidate bioactive agents. A large number of small molecule domains are known, that confer a common function, structure or affinity. In addition, as is appreciated in the art, areas of weak amino acid homology may have strong structural homology.
• a number of these molecules, domains, and/or corresponding consensus sequences are known, including, but are not limited to, SH-2 domains, SH- 3 domains, Pleckstrin, death domains, protease cleavage/recognition sites, enzyme inhibitors, enzyme substrates, Traf, etc.
• nucleic acid binding proteins containing domains suitable for use in the invention.
• leucine zipper consensus sequences are known.
• known peptide ligands can be used as the starting backbone for the generation of the primary library.
• structures known to take on certain conformations may be used to create a backbone, and then sequences screened for those that are likely to take on that conformation.
• ministructures known, sometimes referred to as “presentation structures”, that can confer conformational stability or give a random sequence a conformationally restricted form. Proteins interact with each other largely through conformationally constrained domains. Although small peptides with freely rotating amino and carboxyl termini can have potent functions as is known in the art, the conversion of such peptide structures into pharmacologic agents is difficult due to the inability to predict side-chain positions for peptidomimetic synthesis.
• synthetic presentation structures i e artificial polypeptides
• Preferred presentation structures maximize accessibility to the peptide by presenting it on an exterior loop
• suitable presentation structures include, but are not limited to, mmibody structures, loops on beta-sheet turns and coiled-coil stem structures in which residues not critical to structure are randomized, zinc-finger domains, cysteine-hnked (disulfide) structures, transglutaminase linked structures, cyclic peptides, B- loop structures, helical barrels or bundles, leucme zipper motifs, etc
• the presentation structure is a coiled-coil structure, allowing the presentation of the randomized peptide on an exterior loop
• a coiled-coil structure allowing the presentation of the randomized peptide on an exterior loop
• investigators have isolated peptides capable of high affinity interaction with the appropriate target
• coiled-coil structures allow for between 6 to 20 randomized positions, (see Martin et al , EMBO J 13(22) 5303-5309 (1994), incorporated by reference)
• the presentation structure is a mmibody structure
• a "mmibody” is essentially composed of a minimal antibody complementarity region
• the secondary library generation and creation proceeds as for the known scaffold protein, including recombination of variant positions and/or ammo acid residues, either computationally or experimentally
• libraries of DNA expressing the protein sequences defined by the automated protein design methods can be produced Codons can be randomized at only the nucleotide sequence triplets that define the residue positions specified by the automated design method
• mixtures of base triplets that code for particular ammo acids could be introduced into the DNA synthesis reaction to attach a full triplet defining an ammo acid in one reaction step
• a library of random DNA ohgomers could be designed that biases the desired positions toward certain ammo acids, or that restricts those positions to certain am o acids The ammo acids biased for would be those specified in the virtual screening, or a subset of those Multiple DNA libraries are synthesized that code for different subsets of ammo acids at certain positions, allowing generation of the amio acid diversity
• the random peptide libraries may be done using the frequency tabulation and experimental generation methods including multiplexed PCR, shuffling, etc
• the present invention provides computer readable memories, central processing units, associated circuitry, and other associated compositions to implement the invention
• the apparatus of the invention may include a central processing unit which communicates with a memory and a set of input/output devices (e g , keyboard, mouse, monitor, printer, etc ) 26 through a bus
• a central processing unit which communicates with a memory and a set of input/output devices (e g , keyboard, mouse, monitor, printer, etc ) 26 through a bus
• input/output devices e g , keyboard, mouse, monitor, printer, etc
• the present invention is directed toward the automated protein design program and secondary library generator stored in the memory
• the automated protein design program and/or the secondary library generator may be implemented with a side chain module As discussed in detail in the associated applications, the side chain module establishes a group of potential rotamers for a selected protein backbone structure
• the protein design program may also be implemented with a ranking module As discussed in detail below, the ranking module analyzes the interaction of rotamers with the protein backbone structure to generate optimized protein sequences
• the protein design program may also include a search module to execute a search, for example a Monte Carlo search as described below, in relation to the optimized protein sequences
• an assessment module may also be used to assess physical parameters associated with the derived proteins, as discussed further below
• the memory also stores a protein backbone structure, which is downloaded by a user through the input/output devices
• the memory also stores information on potential rotamers derived by the side chain module
• the memory stores protein sequences generated by the ranking module The protein sequences may be passed as output to the input/output devices
• the expression vectors may be either self-rephcating extrachromosomal vectors or vectors which integrate into a host genome Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the library protein.
• control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
• the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
• Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
• transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the library protein, as will be appreciated by those in the art; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the library protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
• the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
• the regulatory sequences include a promoter and transcriptional start and stop sequences.
• Promoter sequences include constitutive and inducible promoter sequences.
• the promoters may be either naturally occurring promoters, hybrid or synthetic promoters.
• Hybrid promoters which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
• the expression vector may comprise additional elements.
• the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification
• the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct
• the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector Constructs for integrating vectors and appropriate selection and screening protocols are well known in the art and are described m e g , Mansour et al , Cell, 51 503 (1988) and Murray, Gene Transfer and Expression Protocols, Methods in Molecular Biology, Vol 7 (Clifton Humana Press, 1991 )
• the expression vector contains a selection gene to allow the selection of transformed host cells containing the expression vector, and particularly in the case of mammalian cells, ensures the stability of the vector, since cells which do not contain the vector will generally die Selection genes are well known in the art and will vary with the host cell used
• selection gene herein is meant any gene which encodes a gene product that confers resistance to a selection agent Suitable selection agents include, but are not limited to, neomycin (or its analog G418), blasticidin S, histinidol D, bleomycin, puromycm, hygromycm B, and other drugs
• the expression vector contains a RNA splicing sequence upstream or downstream of the gene to be expressed in order to increase the level of gene expression See Barret et al , Nucleic Acids Res 1991 , Groos et al , Mol Cell Biol 1987, and Budiman et al , Mol Cell Biol 1988
• a preferred expression vector system is a retroviral vector system such as is generally described in Mann et al , Cell, 33 153-9 (1993), Pear et al , Proc Natl Acad Sci U S A , 90(18) 8392-6 (1993),
• Kitamura et al Proc Natl Acad Sci U S A , 92 9146-50 (1995), Kmsella et al , Human Gene Therapy, 7 1405-13, Hofmann et al ,Proc Natl Acad Sci U S A , 93 5185-90, Choate et al , Human Gene Therapy, 7 2247 (1996), PCT/US97/01019 and PCT/US97/01048, and references cited therein, all of which are hereby expressly incorporated by reference
• the library proteins of the present invention are produced by cultu ⁇ ng a host cell transformed with nucleic acid, preferably an expression vector, containing nucleic acid encoding an library protein, under the appropriate conditions to induce or cause expression of the library protein
• the conditions appropriate for library protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation
• the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction
• the timing of the harvest is important
• the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield
• the type of cells used in the present invention can vary widely Basically, a wide variety of appropriate host cells can be used, including yeast, bacteria, archaebactena, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkat cells, mast cells and other endocrine and exoc ⁇ ne cells, and neuronal cells See the ATCC cell line catalog, hereby expressly incorporated by reference
• the expression of the secondary libraries in phage display systems are particularly preferred, especially when the secondary library comprises random peptides
• the cells may be genetically
• the library proteins are expressed in mammalian cells Any mammalian cells may be used, with mouse, rat, primate and human cells being particularly preferred, although as will be appreciated by those in the art, modifications of the system by pseudotyping allows all eukaryotic cells to be used, preferably higher eukaryotes
• a screen will be set up such that the cells exhibit a selectable phenotype in the presence of a random library member
• cell types implicated in a wide variety of disease conditions are particularly useful, so long as a suitable screen may be designed to allow the selection of cells that exhibit an altered phenotype as a consequence of the presence of a library member within the cell
• suitable mammalian cell types include, but are not limited to, tumor cells of all types (particularly melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell) , mast cells, eosmophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratmocytes, melanocytes, liver cells, kidney cells, and adipocytes Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos, etc See
• Mammalian expression systems are also known in the art, and include retroviral systems
• a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for library protein into mRNA
• a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site
• a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box
• An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation
• Examples include the SV40 early promoter, mouse mammary tumor virus
• transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation Examples of transcription terminator and polyadenlytion signals include those derived form SV40
• library proteins are expressed in bacterial systems
• Bacterial expression systems are well known in the art
• a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of library protein into mRNA
• a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site
• Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan Promoters from bacte ⁇ ophage may also be used and are known in the art
• synthetic promoters and hybrid promoters are also useful, for example, the tac promoter is a hybrid of the trp and lac promoter sequences
• a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to
• the ribosome binding site is called the Shme-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon
• the expression vector may also include a signal peptide sequence that provides for secretion of the library protein in bacteria
• the signal sequence typically encodes a signal peptide comprised of hydrophobic ammo acids which direct the secretion of the protein from the cell, as is well known in the art
• the protein is either secreted into the growth media (gram-positive bacteria) or into the pe ⁇ plasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria)
• the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicil n, chloramphenicol, erythromycin, kanamycin, neomycm and tetracyclme Selectable markers also include biosynthetic genes, such as those in the histidme, tryptophan and leucme biosynthetic pathways
• Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E coli, Streptococcus cremons, and
• Streptococcus lividans among others
• the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others
• library proteins are produced in insect cells
• Expression vectors for the transformation of insect cells and in particular, baculovirus-based expression vectors, are well known in the art and are described e g , in O'Reilly et al , Baculovirus Expression Vectors A Laboratory Manual (New York Oxford University Press, 1994)
• library protein is produced in yeast cells
• yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K lactis, Pichia guillenmondii and
• P pasto ⁇ s, Schizosaccharomyces pombe, and Yarrowia lipolytica Preferred promoter sequences for expression in yeast include the inducible GAL1.10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokmase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate- dehydrogenase, hexokmase, phosphofructokmase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene
• Yeast selectable markers include ADE2, HIS4, LEU2, TRP1 , and ALG7, which confers resistance to tunicamycin, the neomycm phosphotransferase gene, which confers resistance to G418, and the CUP1 gene, which allows yeast to grow in the presence of copper ions
• the library protein may also be made as a fusion protein, using techniques well known in the art
• the library protein may be fused to a carrier protein to form an immunogen
• the library protein may be made as a fusion protein to increase expression, or for other reasons
• the library protein is an library peptide
• the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes
• other fusion partners may be used, such as targeting sequences which allow the localization of the library members into a subcellular or extracellular compartment of the cell, rescue sequences or purification tags which allow the purification or isolation of either the library protein or the nucleic acids encoding them, stability sequences, which confer stability or protection from degradation to the library protein or the nucleic acid encoding it, for example resistance to proteolytic degradation, or combinations of these, as well as linker sequences as needed
• suitable targeting sequences include, but are not limited to, binding sequences capable of causing binding of the expression product to a predetermined molecule or class of molecules while retaining bioactivity of the expression product, (for example by using enzyme inhibitor or substrate sequences to target a class of relevant enzymes), sequences signalling selective degradation, of itself or co-bound proteins, and signal sequences capable of constitutively localizing the candidate expression products to a predetermined cellular locale, including a) subcellular locations such as the Golgi, endoplasmic reticulum, nucleus, nucleoh, nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and cellular membrane, and b) extracellular locations via a secretory signal Particularly preferred is localization to either subcellular locations or to the outside of the cell via secretion
• the library member comprises a rescue sequence
• a rescue sequence is a sequence which may be used to purify or isolate either the candidate agent or the nucleic acid encoding it
• peptide rescue sequences include purification sequences such as the H ⁇ s 6 tag for use with Ni affinity columns and epitope tags for detection, immunoprecipitation or FACS (fluoroscence-activated cell sorting)
• Suitable epitope tags include myc (for use with the commercially available 9E10 antibody), the BSP biotmylation target sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST
• the rescue sequence may be a unique ohgonucleotide sequence which serves as a probe target site to allow the quick and easy isolation of the retroviral construct, via PCR, related techniques, or hybridization
• the fusion partner is a stability sequence to confer stability to the library member or the nucleic acid encoding it
• peptides may be stabilized by the incorporation of glycmes after the initiation methionine (MG or MGGO), for protection of the peptide to ubiquitmation as per Varshavsky's N-End Rule, thus conferring long half-life in the cytoplasm
• MG or MGGO initiation methionine
• two pro nes at the C-termmus impart peptides that are largely resistant to carboxypeptidase action
• the presence of two glycmes prior to the prohnes impart both flexibility and prevent structure initiating events in the di-prohne to be propagated into the candidate peptide structure
• preferred stability sequences are as follows MG(X) n GGPP, where X is any ammo acid and n is an integer of at least four
• the library nucleic acids, proteins and antibodies of the invention are labeled By
• labels herein is meant that nucleic acids, proteins and antibodies of the invention have at least one element, isotope or chemical compound attached to enable the detection of nucleic acids, proteins and antibodies of the invention
• labels fall into three classes a) isotopic labels, which may be radioactive or heavy isotopes, b) immune labels, which may be antibodies or antigens, and c) colored or fluorescent dyes The labels may be incorporated into the compound at any position
• the library protein is purified or isolated after expression
• Library proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing
• the library protein may be purified using a standard anti-library antibody column Ultraf ⁇ ltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Sprmger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the library protein In some instances no purification will be necessary Once expressed and purified if necessary, the library proteins and nucleic acids are useful in a number of applications
• the secondary libraries are screened for biological activity These screens will be based on the scaffold protein chosen, as is known in the art Thus, any number of protein activities or attributes may be tested, including its binding to its known binding members (for example, its substrates, if it is an enzyme), activity profiles, stability profiles (pH, thermal, buffer conditions), substrate specificity, immunogenicity, toxicity, etc
• a first plurality of cells is screened That is, the cells into which the library member nucleic acids are introduced are screened for an altered phenotype
• the effect of the library member is seen in the same cells in which it is made, i e an autoc ⁇ ne effect
• a “plurality of cells” herein is meant roughly from about 10 3 cells to 10 8 or 10 9 , with from 10 6 to 10 8 being preferred
• This plurality of cells comprises a cellular library, wherein generally each cell within the library contains a member of the secondary library, i e a different library member, although as will be appreciated by those in the art, some cells within the library may not contain one and and some may contain more than one
• the distribution of library members within the individual cell members of the cellular library may vary widely, as it is generally difficult to control the number of nucleic acids which enter a cell during electroporation, etc
• the library nucleic acids are introduced into a first plurality of cells, and the effect of the library members is screened in a second or third plurality of cells, different from the first plurality of cells, i e generally a different cell type That is, the effect of the library member is due to an extracellular effect on a second cell, i e an endocrine or parac ⁇ ne effect
• the first plurality of cells may be grown in or on one media, and the media is allowed to touch a second plurality of cells, and the effect measured Alternatively, there may be direct contact between the cells
• "contacting" is functional contact, and includes both direct and indirect
• the first plurality of cells may or may not be screened
• the methods of the present invention comprise introducing a molecular library of library members into a plurality of cells, a cellular library The plurality of cells is then screened, as is more fully outlined below, for a cell exhibiting an altered phenotype The altered phenotype is due to the presence of a library member
• altered phenotype or “changed physiology” or other grammatical equivalents herein is meant that the phenotype of the cell is altered in some way, preferably in some detectable and/or measurable way
• a strength of the present invention is the wide variety of cell types and potential phenotypic changes which may be tested using the present methods Accordingly, any phenotypic change which may be observed, detected, or measured may be the basis of the screening methods herein Suitable phenotypic changes include, but are not limited to gross physical changes such as changes in cell morphology, cell growth, cell viability, adhesion to substrates or other cells, and cellular density, changes in the expression of one or more RNAs, proteins, hpids, hormones, cytokmes, or other molecules, changes in the equilibrium state (i e half-life) or one or more RNAs, proteins, pids, hormones, cytokmes, or other molecules, changes in the localization of one or more RNAs,
• the altered phenotype may be detected in a wide variety of ways, and will generally depend and correspond to the phenotype that is being changed Generally, the changed phenotype is detected using, for example microscopic analysis of cell morphology, standard cell viability assays, including both increased cell death and increased cell viability, for example, cells that are now resistant to cell death via virus, bacteria, or bacterial or synthetic toxins, standard labeling assays such as fluorometric indicator assays for the presence or level of a particular cell or molecule, including FACS or other dye staining techniques, biochemical detection of the expression of target compounds after killing the cells, etc
• the altered phenotype is detected in the cell in which the randomized nucleic acid was introduced, in other embodiments, the altered phenotype is detected in a second cell which is responding to some molecular signal from the first cell
• the library member is isolated from the positive cell This may be done in a number of ways
• primers complementary to DNA regions common to the constructs, or to specific components of the library such as a rescue sequence, defined above are used to "rescue" the unique random sequence
• the member is isolated using a rescue sequence
• rescue sequences comprising epitope tags or purification sequences may be used to pull out the library member, using immunopre ⁇ pitation or affinity columns In some instances, this may also pull out things to which the library member binds (for example the primary target molecule) if there is a sufficiently strong binding interaction between the library member and the target molecule
• the peptide may be detected using mass spectroscopy
• the member is resynthesized and reintroduced into the target cells, to verify the effect
• This may be done using retroviruses, or alternatively using fusions to the HIV-1 Tat protein, and analogs and related proteins, which allows very high uptake into target cells See for example, Fawell et al , PNAS USA 91 664 (1994), Frankel et al , Cell 55 1189 (1988), Savion et al , J Biol Chem 256 1149 (1981 ), Derossi et al , J Biol Chem 269 10444 (1994), and Baldin et al , EMBO J 9 1511 (1990), all of which are incorporated by reference
• sequence of the member is used to generate more libraries, as outlined herein
• the library member is used to identify target molecules, i e the molecules with which the member interacts.
• target molecules i e the molecules with which the member interacts
• the screening methods of the present invention may be useful to screen a large number of cell types under a wide variety of conditions
• the host cells are cells that are involved in disease states, and they are tested or screened under conditions that normally result in undesirable consequences on the cells
• the undesirable effect may be reduced or eliminated
• normally desirable consequences may be reduced or eliminated, with an eye towards elucidating the cellular mechanisms associated with the disease state or signalling pathway
• Type 2 solvation was used (Street and Mayo, 1998)
• the non-polar exposure multiplication factor was set to 1 6
• the non-polar burial energy was set to 0 048 kcal/mol/A 2
• the polar hydrogen burial energy was set to 2 0 kcal/mol
• the Dead End Elimination (DEE) optimization method (see reference) was used to find the lowest energy, ground state sequence DEE cutoffs of 50 and 100 kcal/mol were used for singles and doubles energy calculations, respectively
• MC Monte Carlo
• Table 3 Monte Carlo analysis (amino acids and their number of occurrences (for the top 1000 sequences) .
• This probabihtydistnbution was then transformed into a rounded probability distribution (see Table 4)
• a 10% cutoff value was used to round at the designed positions and the wild type am o acids were forced to occur with a probability of at least 10%
• An E was found at position 169156% of the time However, since this position is adjacentto another designed position, 170, its closeness would have required a more complicated ohgonucleotide library design, E was therefore not included for this position when generating the sequence library (only L was used)
• each oligonucleotide was added at a concentrationthat reflected the probabilities in Table 4
• concentration that reflected the probabilities in Table 4
• at position 72 equal amounts of the two oligonucleotideswere added to the pool
• at position 136 twice as much M-containmg oligonucleotidewas added compared to the N-containing ohgonucleotide
• seven times as much D-containmg ohgonucleotide was added compared to the N- containmg ohgonucleotide
• PCR products were purified using a QIAquick PCR Purification Kit (Qiagen), digested with Xhol and
• PCR products containing the library of mutant TEM-1 ⁇ -iactamase genes were then cloned between a promoter and terminator in a kanamycm resistant plasmid and transformed into E coli An equal number of bacteria were then spread onto media containing either kanamycm or ampicil n All transformed colonies will be resistantto kanamycm, but only those with active mutated ⁇ -lactamase genes will grow on ampicillm After overnight incubation, several colonies were observed on both plates, indicating that at least one of the above sequences encodes an active ⁇ -lactamase The number of colonies on the kanamycm plate far outnumbered those on the ampicilhn plate (roughly a 5 1 ratio) suggesting that either some of the sequences destroy activity, or that the PCR introduces errors that yield an inactive or truncated enzyme
• the purified PCR product containing the library of mutated sequences was then hgated into pCR-Xen1 that had previouslybeen digestedwith Xhol and Hindlll and purified
• the ligation reaction was transformed into competent TOP10 coli cells (Invitrogen) After allowing the cells to recover for 1 hour at 37°C, the cells were spread onto LB plates containing the antibiotic cefotaxime at concentrations ranging from 0 1 ⁇ g/ml to 50 ⁇ g/ml and selected for increasing resistance
• Table 1 Probability of ammo acids at the designed positions resulting from the PDA calculation of the wild type (WT) enzyme structure Only am o acids with a probability greater than 1 % are shown

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