Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-09-26
2002-12-31
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S471000, C435S476000, C435S488000, C435S320100, C435S489000, C435S252330, C435S189000, C435S193000, C435S195000, C435S091400, C435S455000, C435S483000, C435S484000, C435S485000, C435S486000, C435S487000, C435S325000, C435S348000, C435S252500, C435S254300, C435S254600, C435S254700
Reexamination Certificate
active
06500644
ABSTRACT:
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
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
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,
(
Borchert Torben Vedel
Ehrlich Stanislas Dusko
Garbell Jason
Guzo David
Lambiris Elias
Novozymes A/S
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