Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-23
2002-04-09
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S440000, C435S471000, C435S455000, C435S468000
Reexamination Certificate
active
06368805
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to optimizing DNA sequences in order to (a) improve the properties of a protein of interest by artificial generation of genetic diversity of genes encoding proteins having a biological activity of interest by the use of the so-called gene- or DNA shuffling technique to create a large library of “genes”, expressing said library of genes in a suitable expression system and screening the expressed proteins in respect of specific characteristics to determine such proteins exhibiting desired properties or (b) improve the properties of regulatory elements such as promoters or terminators by generation of a library of these elements, transforming suitable hosts therewith in operable conjunction with a structural gene, expressing said structural gene and screening for desirable properties in the regulatory element.
BACKGROUND OF THE INVENTION
It is generally found that a protein performing a certain bioactivity exhibits a certain variation between genera and even between members of the same species differences may exist. This variation is of course even more outspoken at the genomic level.
This natural genetic diversity among genes coding for proteins having basically the same bioactivity has been generated in Nature over billions of years and reflects a natural optimization of the proteins coded for in respect of the environment of the organism in question.
In today's society the conditions of life are vastly removed from the natural environment and it has been found that the naturally occurring bioactive molecules are not optimized for the various uses to which they are put by mankind, especially when they are used for industrial purposes.
It has therefore been of interest to industry to identify such bioactive proteins that exhibit optimal properties in respect of the use to which it is intended.
This has for many years been done by screening of natural sources, or by use of mutagenesis. For instance, within the technical field of enzymes for use in e.g. detergents, the washing and/or dishwashing performance of e.g. naturally occurring proteases, lipases, amylases and cellulases have been improved significantly, by in vitro modifications of the enzymes.
In most cases these improvements have been obtained by site-directed mutagenesis resulting in substitution, deletion or insertion of specific amino acid residues which have been chosen either on the basis of their type or on the basis of their location in the secondary or tertiary structure of the mature enzyme (see for instance U.S. Pat. No. 4,518,584).
In this manner the preparation of novel polypeptide variants and mutants, such as novel modified enzymes with altered characteristics, e.g. specific activity, substrate specificity, thermal, pH and salt stability, pH-optimum, pI, K
m
, V
max
etc., has successfully been performed to obtain polypeptides with improved properties.
For instance, within the technical field of enzymes the washing and/or dishwashing performance of e.g. proteases, lipases, amylases and cellulases have been improved significantly.
An alternative general approach for modifying proteins and enzymes has been based on random mutagenesis, for instance, as disclosed in U.S. Pat. No. 4,894,331 and WO 93/01285.
As it is a cumbersome and time consuming process to obtain polypeptide variants or mutants with improved functional properties a few alternative methods for rapid preparation of modified polypeptides have been suggested.
Weber et al., (1983), Nucleic Acids Research, vol. 11, 5661-5661, describes a method for modifying genes by in vivo recombination between two homologous genes. A linear DNA sequence comprising a plasmid vector flanked by a DNA sequence encoding alpha-1 human interferon in the 5′-end and a DNA sequence encoding alpha-2 human interferon in the 3′-end is constructed and transfected into a rec A positive strain of
E. coli.
Recombinants were identified and isolated using a resistance marker.
Pompon et al., (1989), Gene 83, p. 15-24, describes a method for shuffling gene domains of mammalian cytochrome P-450 by in vivo recombination of partially homologous sequehces in
Saccharomyces cerevisiae
by transforming
Saccharomyces cerevisiae
with a linearized plasmid with filled-in ends, and a DNA fragment being partially homologous to the ends of said plasmid.
In WO 97/07205 a method is described whereby polypeptide variants are prepared by shuffling different nucleotide sequences of homologous DNA sequences by in vivo recombination using plasmid DNA as template.
U.S. Pat. No. 5,093,257 (Assignee: Genencor Int. Inc.) discloses a method for producing hybrid polypeptides by in vivo recombination. Hybrid DNA sequences are produced by forming a circular vector comprising a replication sequence, a first DNA sequence encoding the amino-terminal portion of the hybrid polypeptide, a second DNA sequence encoding the carboxy-terminal portion of said hybrid polypeptide. The circular vector is transformed into a rec positive microorganism in which the circular vector is amplified. This results in recombination of said circular vector mediated by the naturally occurring recombination mechanism of the rec positive microorganism, which include prokaryotes such as Bacillus and
E. coli,
and eukaryotes such as
Saccharomyces cerevisiae.
One method for the shuffling of homologous DNA sequences has been described by Stemmer (Stemmer, (1994), Proc. Natl. Acad. Sci. U.S.A., Vol. 91, 10747-10751; Stemmer, (1994), Nature, vol. 370, 389-391). The method concerns shuffling homologous DNA sequences by using in vitro PCR techniques. Positive recombinant genes containing shuffled DNA sequences are selected from a DNA library based on the improved function of the expressed proteins.
The above method is also described in WO 95/22625. WO 95/22625 relates to a method for shuffling of homologous DNA sequences. An important step in the method described in WO 95/22625 is to cleave the homologous template double-stranded polynucleotide into random fragments of a desired size followed by homologously reassembling of the fragments into full-length genes.
A disadvantage inherent to the method of WO 95/22625 is, however, that the diversity generated through that method is limited due to the use of homologous gene sequences (as defined in WO 95/22625).
Another disadvantage in the method of WO 95/22625 lies in the production of the random fragments by the cleavage of the template double-stranded polynucleotide.
A further reference of interest is WO 95/17413 describing a method of gene or DNA shuffling by recombination of specific DNA sequences—so-called design elements (DE)—either by recombination of synthesized double-stranded fragments or recombination of PCR generated sequences to produce so-called functional elements (FE) comprising at least two of the design elements. According to the method described in WO 95/17413 the recombination has to be performed among design elements that have DNA sequences with sufficient sequence homology to enable hybridization of the different sequences to be recombined.
WO 95/17413 therefore also entails the disadvantage that the diversity generated is relatively limited. Furthermore the methods described are time consuming, expensive, and not suited for automation.
Despite the existence of the above methods there is still a need for better iterative in vivo recombination methods for preparing novel polypeptide variants. Such methods should also be capable of being performed in small volumes, and amenable to automation.
Furthermore, there also is a need for methods providing the possibility of being able to shuffle genes with relatively low homology.
SUMMARY OF THE INVENTION
The present invention relates to a method for the construction of a library of recombined polynucleotides from a number of different starting single or double stranded parental DNA templates, wherein said starting single or double stranded parental DNA templates represent discrete points in a population of genes encoding evolutionary or synthetic homologues of a p
Borchert Torben Vedel
Cherry Joel R.
Jesper Vind
Kretzschmar Titus
Gansheroff Lisa
Garbell Jason I.
Ketter James
Lambiris Elias J.
Novozymes A/S
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