Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-30
2001-01-30
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06180341
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The invention relates generally to the field of protein chemistry. More specifically, the invention relates to methods employed in the rapid generation of a large number of protein mutants. In one embodiment, mutant proteins are developed using PCR mutagenesis in combination with in vitro transcription/translation.
B. Related Art
Understanding the chemical basis of protein structure and function is one of the most important goals in biology. Studies over the last decade have attempted to determine how protein structure dictates biological function. Site-specific mutagenesis has been a powerful tool in these studies. Structure-guided, site-specific mutagenesis represents a powerful tool for the dissection and engineering of protein-ligand interactions (Wells, 1996; Braisted and Wells, 1996). Typically, residues suspected of contacting the ligand are subjected to a limited set of substitutions with other amino acids and their effects on binding are determined. Several studies, in particular with the T4 lysozyme (Matthews, 1995) and the human growth hormone (hGH)-hGH receptor complex (Wells and deVos, 1993), have combined information derived from crystal structures of a protein with site-specific mutagenesis to analyze the role of contact residues with the ligand. Recently, the role of all the contact residues in the hGH receptor protein was analyzed by replacement with alanines to investigate the effects of side chain interactions without creating large-scale perturbations in protein conformation. (Clackson and Wells, 1995; Cunningham et al., 1989). The results clearly show that out of the thirty-three amino acids in the hGH receptor that are in van der Waals contact with the high affinity binding site of human growth hormone, two amino acids are responsible for over 75% of the binding energy. This result, which could not have been predicted on the basis of the crystal structure of the complex alone, demonstrated that a very small subset of the residues at a protein-ligand interface can be responsible for the majority of binding energy. This study demonstrates the value of mutagenesis at a large number of residues within a protein. However, the study of hGH was limited to the substitution of alanine at a relatively small number of amino acids within the protein, and the substitution of functional residues with alanine, or any other single amino acid, can give misleading results regarding their mechanistic importance (Warren et al., 1996).
In addition to site-specific mutagenesis, libraries generated by random mutagenesis have been employed to reveal principles of protein structure. For example, cassette mutagenesis has been used to probe the “information content” of polypeptide sequences (Reidhaar-Olson and Sauer, 1989; Davidson and Sauer, 1994; Davidson et al., 1995). These studies involve the construction of polypeptide mutants composed of random combinations of selected amino acids. The mutants are then analyzed for their thermal denaturation properties. This study, utilizing existing technology to generate and characterize a relatively large number of mutant proteins, was very labor- and time-intensive.
Thus, the comprehensive understanding of protein function typically involves the construction of hundreds, and possibly thousands of mutants. The logistics of such large scale mutagenesis experiments can be prohibitive. For each mutant protein, using current methodology, the appropriate gene construct must be made, a host organism must be transformed with the DNA, transformants must be selected and screened for expression of the protein, and finally, the host cells must be grown to produce the protein. Using currently available methods, the production of a single mutant polypeptide typically takes a minimum of two weeks of work by experienced personnel. While current methods for site specific mutagenesis may be acceptable in structure-guided studies where only a few amino acid substitutions may be of interest, it is impractical and prohibitively expensive when a hundred or more mutants need to be generated and analyzed.
As a target for mutagenesis studies, attention continues to be focused on antibodies, largely because they define a paradigm of high affinity protein binding and are among the most important classes of commercial protein molecules. Antibodies are nearly ideal reagents for the detection of analytes that are present in minute quantities in highly complex samples, for example soil samples or biological fluids. However, because of inherent limitation of the immune system and of hybridoma technology there are many cases in which it has not been possible to produce monoclonal antibodies with the requisite affinity or specificity for a particular application. Fortunately, recent advances in protein design and characterization techniques have paved the way for the engineering of antibodies with desired functions.
At present, the engineering of antibodies that recognize and bind to antigens with higher affinity, are covalently linked to effector molecules such as toxins, exhibit catalytic activity, or have been modified for better in vivo availability and stability represents an exciting and rapidly evolving field (Morrison, 1992; Lillehoj and Malik 1993). Antibodies with tailored properties hold great promise as pharmaceutical reagents, for bioseparations and, perhaps most importantly, as diagnostic reagents in immunoassays.
Antigen binding is determined primarily, but not exclusively, by amino acid residues in the antibody hypervariable or complementarity determining regions (CDRs) I, II, and III of the heavy (H) and light (L) chains. There is evidence that the antigen binding site exhibits a fair degree of plasticity in that a number of amino acid substitutions are tolerated and occasionally improve affinity (Chen et al., 1995; Short et al., 1995). Studies have also demonstrated that the second shell, as well as contact residues, can play an important role in stabilizing the overall conformation of an antibody binding pocket and in turn affect the affinity and fine specificity of the antibody (Schildbach et al., 1993a, Schildbach et al., 1993b, Schildbach et al., 1994).
One method used for the screening of antibody libraries is phage display (Short et al., 1995). In combination with mutagenesis techniques, phage display has been used to explore the effect of amino acid substitutions on antigen affinity. Such large scale studies can provide important information concerning issues such as: (i) the identity of residues that determine antigen affinity and specificity; (ii) what molecular interactions dominate the energetics of binding; (iii) the molecular basis of affinity maturation (Chen et al., 1995; Brown et al., 1996); and (iv) engineering of antibodies tailored for specific applications in biotechnology (Harrison et al., 1996; Burton and Barbas, 1994).
However, while phage display technology can succeed in the identification of sequence motifs that result in a high affinity towards a desired antigen, this approach complements, and does not substitute for, site-specific mutagenesis. First, since only the amino acid(s) that are compatible with high affinity binding can be identified in a biopanning experiment, it is not possible to examine the effect of other amino acid substitutions that may result in slightly lower affinity, such as second shell residues. Second, the polypeptide sequences that can be isolated using phage display are limited by biological constraints. If a particular amino acid is incompatible with the biogenesis or the propagation of the bacteriophage particle then, the corresponding clone cannot be isolated. And third, the isolation of antibody mutants exhibiting alterations in fine specificity or small changes in affinity is technically difficult. These limitations are particularly important for saturation mutagenesis experiments where specific residues are replaced with all nineteen amino acids.
In recent years, there has been increased interest in the use of in vitro protein synthesis to p
Burks Elizabeth A.
Georgiou George
Iverson Brent L.
Board of Regents, The Universiry of Texas System
Fulbright & Jaworski LLP
Patterson Jr. Charles L.
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