Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-06
2002-12-17
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C514S04400A
Reexamination Certificate
active
06495321
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for in vitro molecular evolution of protein function, in particular by shuffling of DNA segments obtained using an exonuclease.
BACKGROUND OF THE INVENTION
Protein function can be modified and improved in vitro by a variety of methods, including site directed mutagenesis (Alber et al, Nature, 5; 330(6143):41-46, 1987) combinatorial cloning (Huse et al, Science, 246:1275-1281, 1989; Marks et al, Biotechnology, 10: 779-783, 1992) and random mutagenesis combined with appropriate selection systems (Barbas et al, PNAS. USA, 89: 4457-4461, 1992).
The method of random mutagenesis together with selection has been used in a number of cases to improve protein function and two different strategies exist. Firstly, randomisation of the entire gene sequence in combination with the selection of a variant (mutant) protein with the desired characteristics, followed by a new round of random mutagenesis and selection. This method can then be repeated until a protein variant is found which is considered optimal (Schier R. et al, J. Mol. Biol. 1996 263 (4): 551-567). Here, the traditional route to introduce mutations is by error prone PCR (Leung et al, Technique, 1: 11-15, 1989) with a mutation rate of ≈0.7%. Secondly, defined regions of the gene can be mutagenized with degenerate primers, which allows for mutation rates up to 100% (Griffiths et al, EMBO. J, 13: 3245-3260, 1994; Yang et al, J. Mol. Biol. 254: 392-403, 1995). The higher the mutation rate used, the more limited the region of the gene that can be subjected to mutations.
Random mutation has been used extensively in the field of antibody engineering. In vivo formed antibody genes can be cloned in vitro (Larrick et al, Biochem. Biophys. Res. Commun. 160: 1250-1256, 1989) and random combinations of the genes encoding the variable heavy and light genes can be subjected to selection (Marks et al, Biotechnology, 10: 779-783, 1992). Functional antibody fragments selected can be further improved using random mutagenesis and additional rounds of selections (Schier R. et al, J. Mol. Biol. 1996 263 (4): 551-567) .
The strategy of random mutagenesis is followed by selection. Variants with interesting characteristics can be selected and the mutated DNA regions from different variants, each with interesting characteristics, are combined into one coding sequence (Yang et al, J. Mol. Biol. 254: 392-403, 1995). This is a multi-step sequential process, and potential synergistic effects of different mutations in different regions can be lost, since they are not subjected to selection in combination. Thus, these two strategies do not include simultaneous mutagenesis of defined regions and selection of a combination of these regions. Another process involves combinatorial pairing of genes which can be used to improve eg antibody affinity (Marks et al, Biotechnology, 10: 779-783, 1992). Here, the three CDR-regions in each variable gene are fixed and this technology does not allow for shuffling of individual gene segments in the gene for the variable domain, for example, including the CDR regions, between clones.
The concept of DNA shuffling (Stemmer, Nature 370: 389-391, 1994) utilizes random fragmentation of DNA and assembly of fragments into a functional coding sequence. In this process it is possible to introduce chemically synthesized DNA sequences and in this way target variation to defined places in the gene which DNA sequence is known (Crameri et al, Biotechniques, 18: 194-196, 1995). In theory, it is also possible to shuffle DNA between any clones. However, if the resulting shuffled gene is to be functional with respect to expression and activity, the clones to be shuffled have to be related or even identical with the exception of a low level of random mutations. DNA shuffling between genetically different clones will generally produce non-functional genes.
Selection of functional proteins from molecular libraries has been revolutionized by the development of the phage display technology (Parmley et al, Gene, 73: 305-391 1988; McCafferty et al, Nature, 348: 552-554, 1990; Barbas et al, PNAS. USA, 88: 7978-7982, 1991). Here, the phenotype (protein) is directly linked to its corresponding genotype (DNA) and this allows for directly cloning of the genetic material which can then be subjected to further modifications in order to improve protein function. Phage display has been used to clone functional binders from a variety of molecular libraries with up to 10
11
transformants in size (Griffiths et al, EMBO. J. 13: 3245-3260, 1994). Thus, phage display can be used to directly clone functional binders from molecular libraries, and can also be used to improve further the clones originally selected.
Random combination of DNA from different mutated clones in combination with selection of desired function is a more efficient way to search through sequence space as compared to sequential selection and combination of selected clones.
SUMMARY OF THE INVENTION
According to one aspect of the present invention, there is provided a method for generating a polynucleotide sequence or population of sequences from a parent polynucleotide sequence encoding one or more protein motifs, comprising the steps of
a) digesting the parent polynucleotide sequence with an exonuclease to generate a population of fragments;
b) contacting said fragments with a template polynucleotide sequence under annealing conditions;
c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotide.
The parent polynucleotide is preferably double-stranded and the method further comprises the step of generating single-stranded polynucleotide sequence from said double-stranded fragments prior to step b). Further, the template polynucleotide is preferably the parent polynucleotide sequence or at least a polynucleotide sequence having sequence in common with the parent nucleotide sequence so that the fragments will hybridize with the template under annealing conditions. For example, if the parent polynucleotide is an antibody, the template may be a different antibody having constant domains or framework regions in common.
Therefore, typically, there is provided a method of combining polynucleotide fragments to generate a polynucleotide sequence or population of sequences of desired characteristics, which method comprises the steps of:
(a) digesting a linear parent double-stranded polynucleotide encoding one or more protein motifs with an exonuclease to generate a population of double stranded fragments of varying lengths;
(b) obtaining single-stranded polynucleotides from said double-stranded fragments; and
(c) assembling a polynucleotide sequence from the sequences derived from step (b).
Preferably the method further comprises the step of (d) expressing the resulting protein encoded by the assembled polynucleotide sequence and screening the protein for desired characteristics.
Prior to assembling the polynucleotide sequence in step (c) the double stranded sequences are preferably purified and then mixed in order to facilitate assembly. By controlling the reaction time of the exonuclease the size of the polynucleotide fragments may be determined. Determining the lengths of the polynucleotide fragments in this way avoids the necessity of having to provide a further step such as purifying the fragments of desired length from a gel.
Further, as some exonuclease digests polynucleotide sequences from both the 3′ and the 5′ ends, the fragments selected may center around the middle of the gene sequence if this particular region of sequence is desired. This sequence from the middle of a gene may be mutated randomly by, for example, error prone PCR and desirable for the shuffling process.
However, in some cases it may be desirable not to shuffle the sequence from the middle of the gene. This may be prevented by choosing long fragme
Borrebaeck Carl Arne Krister
Ottosson Rebecka Ingrid Camilla
Soderlind Ulf Hans Eskil
BioInvent International AB
Dann Dorfman Herrell and Skillman
Horlick Kenneth R.
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