Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-17
2001-09-18
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S069100, C435S463000, C435S471000
Reexamination Certificate
active
06291165
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a new method of shuffling especially heterologous polynucleotide sequences, screening and/or selection of new recombinant proteins resulting therefrom having a desired biological activity, and especially to production and identification of novel proteases exhibiting desired properties.
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 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 optimisation of the proteins coded for in respect of the environment of the organism in question.
However in general 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).
PRIOR ART
Numerous methods to create genetic diversity, such as by site directed or random mutagenesis, have been proposed and described in both the scientific literature and in patent applications. For further details in this respect reference is made to the related art section of WO 95/22625, wherein a review is provided.
One method for the shuffling of homologous DNA sequences has been described by Stemmer (Stemmer, (1994), Proc. Natl. Acad. Sci. USA, 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.
WO 95/22625 is believed to be the most pertinent reference in relation to the present invention in its “gene shuffling” aspect. 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 DNA sequences either by recombination of synthesized double-stranded fragments or recombination of PCR generated sequences. According to the method described in WO 95/17413 the recombination has to be performed among 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.
The present invention do not contain any steps involving production of random fragments by the cleavage of the template double-stranded polynucleotide, as described in WO 95/22625.
Further WO 95/22625 relates to shuffling of homologous, where the present invention relates to shuffling of heterologous genes.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to avoid the limitation of shuffling only homologous DNA sequences by providing a method to shuffle/recombine heterologous sequences of interest.
The solution is to use at least one “conserved sequence region”, wherein there is sufficient degree of homology between the heterologous sequences to be shuffled, as a “linking point” between said heterologous sequences.
Accordingly, a first aspect of the invention relates to a method for shuffling of heterologous sequences of interest comprising the following steps,
i) identification of at least one conserved region between the heterologous sequences of interest;
ii) generating fragments of each of the heterologous sequences of interest, wherein said fragments comprise the conserved region(s); and
iii) shuffling/recombining said fragments using the conserved region(s) as (a) homologous linking point(s).
In an second aspect the invention relates to a method for producing a shuffled protein having a desired biological activity comprising in addition to the steps of the first aspect the further steps:
iv) expressing the numerous different recombinant proteins encoded by the numerous different shuffled sequences from step iii); and
v) screen or select the numerous different recombinant proteins from step ii) in a suitable screening or selection system for one or more recombinant protein(s) having a desired activity.
The term “conserved region” denotes a sequence region (preferably of at least 10 bp), wherein there is a relatively high sequence identity between said heterologous sequences.
In order for the conserved region to be used as “linking point” between said heterologous sequences, the sequence identity between the heterologous sequences, within said conserved regions, is as high as it enables hybridization of the heterologous sequences using said conserved region as hybridization point (“linking point”).
REFERENCES:
patent: 5605793 (1997-02-01), Stemmer
patent: 5811238 (1998-09-01), Stemmer et al.
patent: 5830721 (1998-11-01), Stemmer et al.
patent: 5837458 (1998-11-01), Minshull et al.
patent: WO 95/17413 (1995-06-01), None
patent: WO 95 22625 (1995-08-01), None
Zhao et al., Nucleic Acids Res. 25(6), 1307-1308, 1997.*
Crameri et al., Nature 391, 288-291, 1998.
Borchert Torben Vedel
Cherry Joel R.
Kretzschmar Titus
Gregg Valeta
Horlick Kenneth R.
Novo Nordisk A S
Zelson Steve T.
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