| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 31.12.02 |
| PATENT TITLE |
Method for in vivo production of a mutant library in cells |
| PATENT ABSTRACT | A method for in vivo production of a library in cells comprising a multitude of mutated genetic elements, wherein an error-prone polymerase is used in each ancestral cell to replicate all or a part of a genetic element independently of the host chromosomal replication machinery. The genetic element comprises i) an origin of replication from which replication is initiated, ii) optionally a genetic marker, e.g. a gene conferring resistance towards an antibiotic, iii) a gene encoding the polypeptide of interest. Also methods for the generation of a DNA sequence encoding a desired variant of a polypeptide of interest, and for the determination of such a DNA sequence are described |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | September 26, 2000 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED | James E. Bailey, Toward a Science of Metabolic Engineering, Science, vol. 252, Jun. 21, 1991 |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method for in vivo production of a library of polynucleotides encoding variant polypeptides of interest, the method comprising inserting into a host cell a genetic element capable of independent replication, wherein the genetic element comprises (i) an origin of replication, (ii) a polynucleotide encoding a polypeptide of interest, and optionally (iii) a genetic marker, wherein an error-prone polymerase initiates replication from the origin of replication (i) to generate a library of polynucleotides encoding variant polypeptides of interest. 2. The method of claim 1, wherein the genetic element further comprises (iv) a polynucleotide encoding an error-prone polymerase. 3. The method of claim 1, further comprising expressing the library of polynucleotides to generate variant polypeptides of interest. 4. The method of claim 1, wherein the genetic element is one of a plasmid, a phagemid, a phage, a virus, a retrovirus, or a retrotransposon. 5. The method of claim 1, wherein the polypeptide of interest is an enzyme. 6. The method of claim 1, wherein the host cell is a prokaryote. 7. The method of claim 1, wherein the host cell is a repair-deficient prokaryote cell that comprises one of mutL, mutS, mutH, or a combination thereof. 8. The method of claim 1, wherein the host cell is a fungus. 9. The method of claim 1, wherein the host cell is a mammalian or insect cell. 10. The method of claim 1, wherein the host cell is repair-deficient. 11. The method of claim 2, wherein the error-prone polymerase is one of DNA pol I, DNA pol II, or reverse transcriptase. 12. The method of claim 4, wherein the plasmid comprises a ColEI origin of replication. 13. The method of claim 11, wherein the error-prone polymerase is selected from the group consisting of E. coli DNA pol I, Bacillus subtilis DNA pol I, HIV reverse transcriptase, T4 DNA polymerase, T7 DNA polymerase, and Phi29 DNA polymerase. 14. The method of claim 5, wherein the enzyme is selected from the group of carbonyl hydrolases, carbohydrases, oxidoreductases, transferases, phytases, ligases, and lyases. 15. The method of claim 6, wherein the prokaryote is E. coli. 16. The method of claim 6, wherein the prokaryote is a Bacillus species. 17. The method of claim 16, wherein the prokaryote is Bacillus lentus, Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus subtilis. 18. The method of claim 8, wherein the fungus is one of Aspergillus oryzae, Aspergillus niger, Aspergillus awamori, or Fusarium venenatum. 19. The method of claim 8, wherein the fungus is Trichoderma reseei. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to methods for in vivo production of libraries of polypeptide variants, the screening of these variants and selection of those exhibiting desired properties. The invention furthermore relates to methods for producing the desired polypeptide variants. BACKGROUND OF THE INVENTION An increasing number of polypeptides, including enzymes and non-enzymatic proteins, are being produced industrially, for use in various industries, household, food/feed, cosmetics, medicine etc. One of the major sources for these proteins is and have been microorganism found in nature. The classical approach for finding polypeptides with new and special properties, have been to screen wild type organisms present in nature. This has been a very successful way of procuring polypeptides to be used in such diverse areas as the above mentioned applications. However, often it has not been possible to produce such polypeptides in sufficient amounts because the quantities produced in the natural host systems were too minute to allow a production, and even if the cost was no problem, difficulties could be encountered in providing sufficient amounts in relation to the demand (e.g. human growth hormone). Such problems have to a large degree been overcome by the advent of recombinant techniques for the production of polypeptides. In this art polypeptides are produced by the use of biological systems. Genes encoding certain polypeptides are cloned and transferred into cells that will produce the polypeptides in quantities much larger than those, wherein they are produced in the original organism. Over the latest twenty years a large number of methods for the production of polypeptides according to such techniques have been developed. Often, proteins from natural sources do not meet the requirements for certain applications, and it will be necessary to modify existing proteins towards certain activities or biophysical properties. It is possible to generate new variants of a protein by classical mutagenesis of the microorganism using radiation (X-ray and UV) or chemical mutagens. However, since this approach is a very labour and time consuming process, in the same last two decades researchers have been developing improvements on existing polypeptides by using more specific and selective recombinant techniques, such as protein and genetic engineering for creating artificial diversity. Based upon considerations using knowledge of the structure-function relationships and general protein chemistry, researchers have come a long way in designing polypeptide variants exhibiting improvements in various properties. However, it has also been realised that the various interactions into which polypeptides take part, are so complex that rational design according to such knowledge has serious limitations, and in recent years methods employing random mutagenesis followed by screening of or selection from very large numbers of variants produced therefrom has gained interest. For this purpose a microbial library of mutants is generated for subsequent expression and screening to determine variants possessing the desired properties. Over the years many both in vitro and in vivo DNA mutagenesis techniques for creating high numbers of different variants of polypeptides have been developed. Considering the fact that a typical naturally occurring polypeptide consists of between 100 and 1000 amino acids, and each may be varied in 20 ways (only to stay within the naturally occurring amino acids), the number of possible variants for a specific polypeptide is enormous. Since the main parameter that defines or measures the usefulness of a microbial collection or library used to identify improved variants of polypeptide is the number of different variants, N, which is comprised in the collection, a need for large libraries has emerged. Especially in cases when a powerful selection system is available, the limiting factor for the identification of the desired polypeptide is the size of the library. In in vitro systems the practical, state of art, limit for N is about 10.sup.8. This is mainly due to inefficiency of transformation (introduction of DNA into the cell) of the manipulated DNA into the host organism. This number varies a lot from organism to organism: in the presently best case, E. coli, the usual efficiency of transformation of in vitro manipulated DNA, e.g. a ligation of DNA fragments or chemical treatment of DNA, leads at the most to library sizes up to 10.sup.8 bacteria (Greg Winter, Current methods in Immunology 5: 253-255, 1993). Very few examples of libraries of this size have been reported. In vitro library constructions in other prokaryotes, such as Bacillus sp., Streptococcus sp. or Staphylococcus sp. will for practical reasons be orders of magnitude below this number. Considering eukaryotic hosts such as Saccharomyces cerevisiae or various Aspergillus sp., an even lower number of transformants can be expected from in vitro manipulated DNA. A special case of a large library has been reported based on in vivo recombination between libraries of antibody light and heavy chains based on a specially designed system useful for that particular case (Griffiths, A. D. et al., 1994, EMBO J. 14: 3245-3260). A number of methods are available to generate variants of a polypeptide in microorganisms in vivo, ranging from very simple, such as treating cells with chemical or physical mutagens, to rather complex, relying on cells that contain an error-prone DNA polymerase but lack the mismatch repair system which corrects the errors (Stratagene, XL1-red (mutS, mutD, mutT) Catalog #200129). But these techniques have a major drawback as the mutagenesis is not targeted to a specific part of the genome (coding for the polypeptide of interest) and high frequencies of mutations are generated also in essential genes for the cell as well as in the target gene, resulting in massive cell death, together with a high number of cells, where the mutations do not influence the polypeptide of interest. Such "noise" will limit the accumulation of mutations in the target region. It is therefore the object of the invention to provide an in vivo target region-specific mutagenesis procedure in order to produce very large numbers, N, of polypeptide variants. A second object of the invention relates to the screening or selection of variants with the desired properties, both by existing and future technologies. SUMMARY OF THE INVENTION The present invention therefore relates to a method for in vivo production of a library in cells comprising a multitude of mutated genetic elements, wherein an error-prone polymerase is used in each ancestral cell to replicate all or a part of a genetic element comprising i) an origin of replication from which replication is initiated, ii) optionally a genetic marker, e.g. a gene conferring resistance towards an antibiotic, iii) a gene encoding the polypeptide of interest, independently of the host chromosomal replication machinery. The invention furthermore relates to a method for the generation of a DNA sequence encoding a desired variant of a polypeptide of interest, wherein i) a mutant library is produced by the above method, ii) said library is cultivated under conditions conducive for the expression of said gene of interest to produce polypeptide variants, iii) said variant polypeptides are screened or selected for a desired property, and hosts producing such desired variants identified and isolated, iv) said genetic element in said hosts is sequenced to elucidate the DNA sequence of the mutant gene encoding a desired variant. And a method for the determination of the DNA sequence encoding a desired variant of a polypeptide of interest, wherein (i) a mutant library is produced by the above method, (ii) said library is cultivated under conditions conducive for the expression of said gene of interest to produce variant polypeptides, (iii) said variant polypeptides are screened or selected for a desired property, and hosts producing desired variants identified and isolated, (iv) said genetic element in said hosts is sequenced to elucidate the DNA sequence of the mutant gene encoding a desired variant. The screening of the library or the selection of the variants depends on the specific polypeptide and which properties thereof it is desired to improve and/or retain. It is therefore necessary to set up a screening protocol for each case. Such protocols involving a number of assays are described in the literature (Clackson et al., Nature 352:624-628, 1991, Bryan, P et al., Proteins 1:326-334, 1986). An elegant approach to the combination of the generation of diversity and the selection of variants with the desired properties would be a combination of the in vivo method of the invention for generating the diversity with a phage display system (Greg Winter, Supra). A specific example of a polypeptide of interest is the alkaline proteases used in the detergent industry for the removal of proteinaceous stains from fabric. In that case the screening may be performed in actual detergent compositions to investigate properties such as thermal stability, oxidation stability, storage stability, substrate specificity and affinity, stability to non-aqueous solvents, pH profile, ionic strength dependence, catalytic efficiency, and wash performance. Furthermore the invention relates to a process for the production of a desired polypeptide variant, wherein (i) a DNA sequence encoding a polypeptide of interest that has been determined according to the method above is introduced into a suitable host in a manner whereby it can be expressed in said host, (ii) said host is cultivated under conditions conducive to the expression of said DNA sequence, and (iii) said polypeptide variant is recovered. Methods for the introduction of the DNA sequence selected into suitable host systems are described in, (Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.). It is also within the abilities of the skilled person to select suitable growth media and other conditions for the host system selected that are conducive for the expression of the polypeptide variant od interest. Guidance hereto may f. ex. be found in (Sambrook et al., supra). Also for the recovery of the polypeptide a large number of methods are available for the separation and purification of proteins, e.g. in (Scopes, R. K., Protein Purification (1987), Springer-Verlag) Lastly, the invention relates to the polypeptides produced by the above method. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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