AU769516B2

AU769516B2 - Method for obtaining (in vitro) recombined polynucleotide sequences, sequence banks and resulting sequences

Info

Publication number: AU769516B2
Application number: AU51717/99A
Authority: AU
Inventors: Daniel Dupret; Fabrice Lefevre; Jean-Michel Masson
Original assignee: Proteus SA
Current assignee: Proteus SA
Priority date: 1998-08-12
Filing date: 1999-08-11
Publication date: 2004-01-29
Anticipated expiration: 2019-08-11
Also published as: WO2000009679A1; JP4448619B2; NO20015787L; FR2782323B1; CA2339901A1; ES2253901T3; JP2002538763A; DK1104457T3; IL141373A0; IL141373A; ATE309342T1; CA2339901C; DE69928263T2; EP1104457B1; AU5171799A; AU769516C; FR2782323A1; DE69928263D1; EP1683861A1; EP1104457A1

Abstract

A method (I) for producing recombined polynucleotide sequences from a library of double-stranded polynucleotide sequences is new and comprises fragmenting and denaturing library sequences, hybridizing them with assembly matrices and selecting sequences with requires properties.. Method (I) for producing recombined polynucleotide sequences from a library of double-stranded polynucleotide sequences is new and comprises: (1) fragmenting the library sequences; (2) denaturing the fragments, optionally in the presence of one or more assembly matrices; (3) hybridizing the fragments with one or more assembly matrices, if these were not present in step (b); (4) ligating the fragments to obtain recombined sequences; and (5) selecting recombined sequences having advantageous properties compared with one or more reference sequences. Independent claims are also included for the following: (1) a recombined polynucleotide sequence obtained and selected by method (I); (2) a vector containing the sequence of (1); (3) a host cell transformed with the sequence of (1) or the vector of (2); (4) a protein encoded by the sequence of (1); (5) a library of polynucleotide sequences as in (1), vectors as in (2), cells as in (3) or proteins as in (4).

Description

PROCESS FOR OBTAINING RECOMBINED NUCLEOTIDE SEQUENCES IN VITRO, LIBRARIES OF SEQUENCES AND SEQUENCES THUS

OBTAINED

The present invention relates to a method of obtaining recombined nucleotide sequences in vitro. The invention is particularly aimed at generating and then selecting polynucleotide sequences which are liable to have one or several advantageous properties as compared to the corresponding properties of reference sequences and therefore capable of conferring an improved phenotype and/or of producing improved proteins.

By reference sequence is understood a sequence having properties close to those being sought.

Different techniques have been developed in order to favor the in vitro recombination between different polynucleotide sequences, among which can be more particularly cited DNA-shuffling (12) and StEP both based on the use of PCR.

DNA-Shuffling comprises two steps, the random fragmenting by DNAse I of polynucleotide sequences, and an amplification by PCR in which the fragments previously generated serve as primers. At each step of hybridization, the change of template provokes recombinations at the level of the regions having homologous sequences. A schematic representation of this method is provided in figure 1A The step consists of mixing different polynucleotide sequences containing various mutations in the presence of a pair of pnmers. This mixture is subjected to a PCR type of reaction in which the hybridization and polymerization steps are grouped together in a single step of very short duration. These conditions permit hybridization of primers but reduce the duration hence the length of polymerization, in such a way that the fragments which are partially synthesized are randomly hybridized to the polynucleotide 2 sequences having different mutations, thus permitting the recombination. A schematic representation of this method is provided at Figure lB.

In each of these two methods, the polymerization step is indispensable to the process of recombination.

Thus according to the polymerase chosen, this polymerization step can bring about undesired supplemental mutations. Moreover, the DNA-shuffling and the step procedures rest on the principle of the hybridization of a "mega-primer" to a template, which likely leads to difficulties in implementation for polynucleotide sequences of size greater than 1.5 kbp Finally, these two techniques do not permit the control of the rate of recombination, since they are made randomly in the course of successive polymerization steps.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and eooo S"pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

For the purposes of this specification it will be o clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The present invention provides a simple process for the preparation of recombined polynucleotide H:\RBe11\Keep\51717-99.dOC 31/10/03 3 sequences, permitting the generation of polynucleotide sequences capable of displaying advantageous properties as compared to the corresponding properties of the sequences of reference and therefore capable of conferring an improved phenotype and/or producing improved proteins.

This is achieved by a process of obtaining recombinant polynucleotide sequences in vitro starting from a library of polynucleotide sequences, which includes the following steps: a) preparing a library of double-stranded polynucleotide sequences from a native gene by steps of: i) successive directed mutagenesis; ii) error prone PCR; iii) random chemical mutagenisis; iv) in vivo random mutagenisis; or v) combining genes of close or distinct families within the same or different species in such a way as to arrange a variety of polynucleotide sequences in an initial library; b) generating fragments from the library; c) denaturation of the fragments; d) hybridization of said fragments with one or more assembling templates; e) ligation of said fragments in order to obtain recombined polynucleotide sequences; and f) selection of recombined polynucleotide sequences which have advantageous properties as compared to the corresponding properties of one or more reference sequences.

The process of the invention can include at the output of step and before step the repeating of Hi\RBell\Keep\51717-99.doc 31/10/03 3a steps and with the products of step (d) rather than with the fragments of step This embodiment is particularly useful in the case where, at the outlet of step all the fragments are not ligated. In this case, the process of the invention additionally includes, at the end of step (d) and before step one or several of the following reaction cycles: denaturation of the ligated and non-ligated fragments coming out of step optionally in the presence of one or several assembling templates, hybridization of said fragments with one or several assembling templates if it (they) is (are) not present at the time of the denaturation, -ligation of said fragments.

These denaturation, hybridization and ligation reactions are equivalent in steps and but are carried out with the ligated and non-ligated fragments resulting from step rather than with the fragments of step The process of the invention can moreover include one or several of the following steps: -the separation of the recombined polynucleotide sequences from the assembling template or templates before step the amplification of the double-stranded recombined polynucleotide sequences before step the cloning of the recombined polynucleotide 35 sequences possibly after separation of the **recombined strands from the template or templates and obtaining of the corresponding H:\RBell\Keep\51717-99.doc 31/10/03 3b double strand before step H:\RBeI1\Keep\51717-99.doc 31/10/03 4 The ends of the fragments generated at step are such that there can be adjacent hybridization of these ends to the assembling template or templates at step and ligation of these fragments with each other at step The polynucleotide sequences of the library on which the process of the invention is carried out must include zones of homology either between them or with the assembling templates, so as to permit the generating of the ends of the fragments such as described above.

One advantageous embodiment of the process of the invention io consists in simultaneously carrying out steps and according to a reaction called RLR for the English expression of "Recombining Ligation Reaction." Besides the advantages previously indicated, the process of the invention is notable in that it favors and accelerates the random recombination in vitro of polynucleotide sequences and these polynucleotide sequences can be genes. By gene is understood a DNA fragment or sequence associated with a biological function. A gene can be obtained in different manners, such as chemical synthesis, synthesis by polymerization or by extraction of said gene starting with a source of nucleic acids.

The in vitro recombination of the polynucleotide sequences of the initial library by the process of the invention therefore permits the obtaining of a new library containing sequences having acquired one or several characteristics of the sequences of the previous library. The process of the invention therefore comprises an in vitro technique of evolution.

The process of the invention comprises an alternative to recombinant PCR such as implemented in the techniques of DNA shuffling (12) or of StEP since it does not require the in vitro polymerization step in order to end up with the recombination. To the contrary, the key step of the process of the invention is the step of ligation on an assembling template, which assures a very high degree of faithfulness in the course of the recombination events.

The process of the invention is notable in that it permits a considerable increase in the efficiency of the reassembling of the fragments to ligate. In effect, in the case of a sequence cut up into n fragments, there exists n n possibilities of reassociation of the fragments by using a classical process of ligation (without using a reassembling template which directs the ligation), among which a single form is of interest. In the case of the process of the invention, the ligation is directed by the assembling template, which permits to direct obtention of the single form of interest.

The fragmenting of these polynucleotide sequences at step can be done either in a controlled manner or in a random manner.

In the case of a fragmenting carried out in a controlled manner, the fragmenting permits controlling with precision the degree of desired recombination and the position of the points of recombination. According to a preferred embodiment of the process of the invention, step consists of subjecting the polynucleotide sequences of the library to hydrolysis by the action of one or several restriction enzymes. In this way, in one particular embodiment of the process of the invention, the degree of recombination and the position of the points of recombination of the recombined polynucleotide sequences are determined by the fragmenting of step Thus, the larger the number of fragments generated by the sequence, the higher the number of fragments necessary to recompose a sequence, which leads to a higher rate of recombination. Moreover, the nature and the position of the ends of the fragments generated in this embodiment of the process of the invention can be known and controlled, which permits: controlling with precision the zones where the recombination takes place, or inducing recombination between polynucleotide sequences, for example genes, if the ends of the fragments are created in zones of homology between these sequences, or in zones of homology between these sequences and the assembling template or templates.

6 In the case of a random fragmenting, any enzymatic or mechanical means known to a person skilled in the art allowing a random cutting of the double-stranded DNA can be used, such as for example DNAse I digestion or sonication.

The process of the invention, which permits a considerable increase in the efficiency of reassembling fragments to be ligated, can therefore be applied to the directing of blunt end multi-molecular ligation. In this 0o application, single or double-stranded oligonucleotides complementary only to the 3' end of a fragment and 5' of the adjacent fragment are used as the assembling template in steps or which permits adjacent hybridization of these two ends on the same template after the denaturation step. Once hybridized, the ends of the fragments can be ligated so as to direct the direction of the ligation of the blunt ended fragments The same approach can be contemplated for the positioning of the ligation of fragments having sticky ends.

An especially preferred embodiment of the process of the invention consists of adding at step and/or at step enzymes capable of recognizing and of cutting in a specific manner the non-hybridized ends of fragments, when these overlap with other hybridized fragments on the same template. A preferred example of this type of enzyme is the Flap endonuclease enzyme A particular embodiment of the process of the invention therefore consists of using enzymes of the Flap endonuclease type when the fragments generated at step can be overlapping during the hybridization on the assembling template (of) at step In this way, during the hybridization of single stranded DNA fragments on a template, these enzymes have the property of recognizing and of cutting, in a specific manner, the non-hybridized ends of these fragments, when they overlap with other hybridized fragments on the same template. In the course 7 of the hybridization step these enzymes therefore permit increasing the number of adjacent ends that can be ligated at step which is particularly important in the case of fragments obtained by random cutting, as these fragments have zones overlapping with each other when they are hybridized on the assembling template.

In a particular embodiment of the process of the invention using a ligase active at high temperature and preferably thermostable at step the endonucleases, capable of recognizing and of cutting in a specific manner the to non-hybridized ends of the fragments such as the Flaps, added at step (c) and/or at step will have the same properties of thermoresistance and of high temperature activity as said ligase.

The library of polynucleotide sequences on which the process of the present invention is carried out can be generated by any method known to a person skilled in the art, for example starting with a wild type gene, by successive steps of directed mutagenesis, by "error prone" PCR by random chemical mutagenesis, by random in vivo mutagenesis, or by combining genes of close or distinct families within the same species or different species so as to obtain a variety of polynucleotide sequences in said library.

Among these techniques, the invention more particularly contemplates a process in which the library of double-stranded polynucleotide sequences is obtained by a polymerization chain reaction carried out under conditions that permit creation of random point mutations.

The initial library of double-stranded polynucleotide sequences can be composed of synthetic sequences which will be fragmented at step or which can be the fragments of step According to a preferred embodiment for carrying out the process of the invention, step consists of subjecting the polynucleotide sequences of the library to hydrolysis by the action of one or several restriction enzymes.

8 In order to increase the degree of recombination generated by the process of the invention, it is sufficient to increase the number of restriction fragments by using restriction enzymes having a large number of cutting sites on the polynucleotide sequences of the library, or by combining several restriction enzymes. In the case of using a thermostable and thermoactive ligase, the size of the smallest fragment thus generated will advantageously be greater or equal to 40 bp, in order to retain a hybridization temperature compatible with that of the ligation step which is generally of the order of 65 0

C.

Step can also be carried out by generating a library of fragments by random enzymatic or mechanical treatment. In particular, step can consist of a random treatment with DNAse I of a library of partially heterologous double-stranded polynucleotide sequences. In the case where a random enzymatic or mechanical fragmenting is used in step this embodiment of the process of the invention has the characteristic of permitting the use of fragments generated by this treatment as templates for each other, for the hybridization in the course of step or of the RLR reaction of the steps (c) and simultaneously.

Step can be carried out by combining at least two distinct libraries of fragments separately generated at step starting from the same initial library by different treatments, as for example with different restriction enzymes. In the case of the embodiment of such libraries, the fragments obtained at step are used as templates for each other, for the hybridization during the course of step or the RLR reaction of steps and (d) simultaneously.

The fragments of step of the process of the invention can be equally generated by amplification reactions such as PCR realized on the polynucleotide sequences of the library. Two solutions are contemplated. In a first case, the oligonucleotide primers can be conceived in a manner so as 9 to generate fragments having ends which are adjacent all along the length of the assembling sequence. In a second case, the oligonucleotide primers are conceived in a fashion so as to generate fragments having common sequences, these fragments being capable of serving as an assembling template for each other at step or at step The process of the invention permits the combining in a random manner of different fragments obtained at step and of reassembling them during steps and within a polynucloetide sequence. This process therefore reproduces in vitro the recombination phenomena which can occur in vivo by favoring them. The io process of the invention is therefore particularly of interest above all for recombining polynucleotide sequences among themselves in order to generate new polynucleotide sequences having interesting properties as compared to the corresponding properties of the reference sequences.

is The effectiveness of the recombination of the process of the invention depends on the number of fragments generated by a polynucleotide sequence at step Consequently, the process of the invention will use polynucleotide sequences having been fragmented into n fragments, n advantageously being greater or equal to three.

The assembling template of step or is for example a polynucleotide sequence coming from the initial library or a consensus sequence of said library, single or double-stranded. In the case where the assembling template is incorporated directly at step of the invention, this template must be single-stranded.

According to a variant of the process of the invention, the assembling templates of steps or are composed of single or double-stranded oligonucleotides.

According to a particular embodiment of the process of the invention, oligonucleotides, single or double-stranded, of variable length, are added at step or in addition to the template. These oligonucleotides are designed (to be capable of being) so as to be substituted for a portion of the fragments of step in effect, their sequence is such that: if they are perfectly homologous with the sequence of the s fragment which they are replacing, they favor certain combinations, or if they are partially heterologous with the sequence of the fragment that they are replacing, they introduce one or a more directed supplemental mutations.

Before step of the process of the invention, it is possible to separate the recombined polynucleotide sequences from the assembling template thanks to a labeling present in the assembling template or in the recombined polynucleotide sequences. It is in effect possible to label each strand of the is template according to techniques known to a person skilled in the art. For example, the label of the assembling template can be a hapten and the recombined polynucleotide sequences templateare separated from the assembling template by techniques known to a person skilled in the art, such as for example an anti-hapten antibody boundon a support or a biotinstreptavidine reaction, if the hapten is a biotin label.

Other techniques can be employed in order to separate the recombined polynucleotide sequences from the assembling template. The assembling template can also be prepared specifically in a way so as to facilitate its elimination at the end of the process of the invention. It can thus be synthesized by PCR amplification using methylated dATP, which permits its degradation by the restriction endonuclease Dpn I. In this case, the recombined polynucleotide sequences must not contain methylated dATP.

The template can also have been prepared by PCR amplification by using 3o dUTP, which permits its degradation by treatment with a uracyl-DNAglycosylase. Conversely, it is possible to protect the recombined polynucleotide sequences by amplifying them by selective PCR with oligonucleotides having 5' phosphorothioate groups. A treatment with an exonuclease then permits the specific digestionof the assembling template.

11 The process of the invention can include before the possible cloning of step a step of amplification of the recombined polynucleotide sequences.

Any amplification technique is acceptable, notably a PCR amplification. One of the most simple consists of carrying out a PCR which permits specific amplification of the recombined polynucleotide sequences owing to primers which can only be hybridized at the ends of the recombined sequences. The PCR products are then cloned in order to be characterized and the polynucleotide sequences having advantageous properties as compared to o1 the corresponding properties of the reference sequences are selected.

The invention has for its object the generation of polynucleotide sequences liable to have advantageous properties as compared to the corresponding properties of reference sequences. The recombined polynucleotide sequences obtained at step and possibly cloned are screened by any appropriate means in order to select the recombined polynucleotide sequences or the clones having advantageous properties as compared to the corresponding properties of the reference sequences By advantageous property is understood to be, for example, the thermostability of an enzyme or its abilityto function under conditions of pH or of temperature or of saline concentration more adapted to an enzymatic process than the control proteins usually used for said process. Forexample, such a process can be an industrial process to breakdown textile fibers or bleaching paper pulps or producing flavors in the dairy industry, the processes of biocatalysis for the synthesis by an enzymatic pathway of new therapeutic molecules, etc.

According to an advantageous embodiment of the process of the invention, the polynucleotide sequence library can therefore be the result of a screening having permitted selection by any appropriate means of polynucleotide sequences having advantageous properties as compared to control sequences. The sequences selected in this way comprise a limited library.

But, it is also possible to start from a non-limited library in order to preserve the representation of the properties contained in that library.

The sequences coding for the protein or proteins having one or more advantageous properties as compared to the reference proteins are thus selected, by in vivo or in vitro screenings, and can be used to form a new library for a possible repeating of the process of the invention. One advantageous embodiment of the process of the invention therefore consists of using as the library several polynucleotide sequences selected after a first io implementation of the process of the invention, possibly mixed with other polynucleotide sequences. Among the screening techniques which can be applied to each of the clones of step the screening techniques in vitro give rise to the advantage of being free of problems of cellular physiology, and of any drawbacks tied to the in vivo expression cloning. Moreover, this type of screening is easily automated, which permits screening a higher number of recombined polynucleotide sequences.

The invention also relates to a recombined polynucleotide sequence obtained by a process according to the invention, as well as to a vector containing such a recombined polynucleotide sequence, a cellular host transformed by a recombined polynucleotide sequence or by a vector of the invention, as well as a protein coded by this recombined polynucleotide sequence. The invention includes as well the corresponding libraries of recombined polynucleotide sequences, vectors, cellular hosts, or proteins.

Other advantages and features of the invention will appear from the examples of carrying out the invention, which follow and which are referred to in the attached drawings in which: Figure 1 is a schematic representation of the processes of the prior art corresponding respectively to DNA-shuffling (figure 1A) and to StEP (figure 1B).

13 Figure 2 is a schematic representation of an example of carrying out the process of the invention and of certain of its variations and applications.

Figure 3 represents the positions of the ten zones of mutations (Pvu II and Pst I) carried by each mutant of the ponB gene used for the examples of the implementation of the invention.

Figure 4 represents the position of the primers used as compared to the sequence of the ponB gene.

Figure 5 represents the migration on agarose gel of RLR and of PCR reaction products of these RLR reactions.

Figure 6 represents the position of the mutations as compared to the restriction fragments.

I- EXAMPLE The process of the invention was put into practice starting from a library of gene mutants of ponB, coding for the PBP1b of E. coli Ten mutants of this gene were used. The gene sequence of each mutant differs from that of the native gene by a non homologous zone of thirteen to sixteen bases in length resulting from the substitution of five initial codons by five alanine codons according to the technique described by Lefevre et al. The substitution carried by each mutant is characterized by the presence of a unique site of the restriction enzyme Pvu II surrounded by two Pst I enzyme sites, which permits the mutants to be distinguished from each other by their digestion profile with these restriction endonucleases. Figure 3 represents the positions of the ten zones of mutations (Pvu II and Pst I) carried by each mutant.

After PCR amplification of the genes of the ten mutants, the PCR products were purified, mixed in equimolar quantity in order to form the library.

The polynucleotide sequences of this library were digested with the restriction enzymes Hinf I and Bsa I, in such a way as to generate libraries of restriction fragments. The restriction fragments were then incubated with various amounts of the wild type template, at different quantities, in the presence of a thermostable ligase. After several denaturation/hybridization/ligation cycles, a fraction of the reaction mixture was used to carry out a PCR amplification with a couple of primers specific to the 5' and 3' ends of the genes of the mutants and non-specific to the 5' and 3' ends of the wild type template. The amplification product was cloned and the obtained clones were analyzed for their digestion profile with the Pvu II or Pst I restriction endonucleases. The obtained profiles permitted the determination of which fragment(s) of the mutants had been able to be recombined with the others in order to form an entire gene.

II MATERIALS 1) Strains and plasmids The strain MC1061 (F araD139, A (ara-leu)7696, galE15, gaiK16, A (lac)X74, rpsL (StrR), mcrA mcrB1, hsdR2 (rk mk)) is derived from Escherichia coli K12.

The vector pARAPONB stems from the vector pARA13 in which the ponB gene carrying a thrombin-cutting site was introduced between the restriction sites Nco I and Nar I. The vector pET26b+ is one of the pET vectors developed by Studier and Moffatt (13) and commercialized by NOVAGEN Corporation.

2) Oliqonucleotides The oligonucleotides were synthesized by ISOPRIM corporation (Toulouse). The oligonucleotide sequences are reported in Table I below.

Table I Oligo N 5' ACTGACTACCATGGCCGGGAATGACCGCGAGCC 3' Oli go E 5' CCGCGGTGGAGCGAATTCTAATTACTACCAAACATATCC 3' Oligo Ml 5' GCGCCTGAATATTGCGGAGAAAAAGC 3' Oligo M2 5' ACAACCAGATGAAAAGAAAGGGTTAATATC 3 Oligo Al 5' ACTGACTACCATGGCC 3' Oligo A2 5' CCGCGGTGGAGCGAA1TC 3' 3) Reagents The restriction and modification enzymes cited in Table 11 below were used according to the recommendations of the suppliers- Table 11 Enzyme Concentration Supplier NlcoI 10 U41 New England Biolabs PstI 20 U/l.I New England Biolabs Eco RI 20 u/141 New England Biolabs Bsa 1 5 U/MIl New England Biolabs Hinf 1 10 U/ L4 New England Biolabs PvU I 10 U/±Il New England Biolabs T4 DNA ligase 400 U/MIL New England 13olabs Taq DNA polymerase 5 U/MI PROM EGA AMPLIGASE 100 U/PI EPICENTRE The buffers used are reported in Table Ill below Restriction A 10X 500 mM NaCI, 100 mM Tris HCI pH 7.9, 100mM MgCI 2 10mM DTT Restriction B 10X 1 M NaCI, 500 mM Tris HCI pH 7.9, 100 mM MgCI 2 mM DTT Restriction C 10X 500 mM NaCI. 1 M Tris HCI pH 7.5, 100 mM mM MgCI 2 0.25% TRITON X100® AMPLIGASE 10X 200 mM Tris HCI pH 8.3, 250 mM KCI, 100 mM MgCl 2 5 mM NAD, 0.1% TRITON X100® Ligation 10X 500 mM Tris HCI pH 7.5, 100 mM MgCl2, 100 mM DTT, 10 mM ATP, 2509/ml BSA III PREPARATION OF THE TEMPLATE The wild type ponB gene was amplified by a PCR reaction step by using as primers the oligonucleotides M1 and M2 (Fig. Five PCR reactions were prepared by adding 50 ng of pPONBPBR plasmid carrying the wild type gene to a mixture containing 10 gil of polymerization buffer, 10 Fl of dNTPs 2mM, 20 pmol of each oligonucleotide M1 and M2, and 5U of Taq DNA polymerase, in a final volume of 100 pl. These mixtures were incubated to in Perkin-Elmer 9600 Thermocycler according to the following program: (94 OC 2 min.) (94 0 C 15 sec. 60 0 C 30 sec. -72 0 C 1 min.) x 29 cycles (72"C -3 min.).

The product of the five PCR was mixed and loadedon a 1% TBEagarose gelAfter migration and staining of the gel with ethidium bromide, the band at 2651 bp, corresponding to the ponB gene amplification product surrounded by two fragments of 26 bp and 90 bp respectively, was visualized by trans-illumination under ultraviolet, and cut out with a scalpel in order to be purified with the QUIAquick system (QIAGEN). Allthe DNA thus purified was eluted in 120 pl of buffer T. The concentration of this DNA was approximatively 100 ng/pl as measured by its absorbanceat 260 nm IV PREPARATION OF THE LIBRARY 1) Amplification of the mutant genes The genes of the ten mutants were separately amplified by a PCR reaction witholigonucleotides N and E. These oligonucleotides introduce respectively the restriction sites Nco I and Eco RI, permitting the cloning of the products obtained with these two sites.

Each PCR reaction was prepared by adding 50 ng of the plasmid o1 carrying the mutant gene to a mixture containing 10 |l of polymerization buffer, 10 pl of dNTPs 2mM, 20 pmol of each oligonucleotide N and E, and of Taq DNA polymerase, in a final volume of 100 41. This mixture was incubated in a Perkin-Elmer 9600 thermocycler according to the following program: (94 0 C 2 min.) (94 0 C 15 sec. 60 0 C 30 sec. 72 0 C 1 min.) x 29 cycles (72 0 C 3 min.).

The specificity of the genetic amplification was verified by restriction profile with the Pvu II endonuclease, by incubating 5 Jp of each PCR product 1 hour at 37 OC in a mixture containing 3 1 l of restriction buffer A and 5U of the Pvu IlIenzyme in a final volume of 30 pl. 15 tl of that digestion reaction were loaded on a TBE 1% agarose gel. After migration and staining with ethidium bromide, the gel was exposed to ultraviolet. The visualization of the restriction fragments permitted confirmation of the specificity of the genetic amplification of each mutant gene.

In parallel, 3 41 of each PCR reaction were loaded on a TBE 1% agarose gel. After migration, the gel was treated as above. The intensity of each band permitted the assessment that the genetic amplifications had the same yield.

2) Creation of libraries of restriction fragments.

41 of each of the ten PCR were mixed and loaded on a 1% TBE agarose gel. After migration and staining with ethidium bromide, the band at 2572 bp, corresponding to the amplification product of the genes of the ten mutants, was cut out with a scalpel and purified with the Quiaquick system (QIAGEN).

Allthe DNA thus purified was eluted in 120 .1 of buffer T. The concentration of this DNA was approximately 100 ng/pl according to its absorbance at 260 nm.

In order to generate the libraries of restriction fragments, 100 1 pl of this DNA were incubated for one hour at 50°C in a mixture containing 12 )l of 0o restriction buffer B, 1.2 pI of BSA (at 10 mg/ml), 25 U of the enzyme Bsa I and 4 p1 of water. Then, 2 p of restriction buffer B, 2 pl of BSA (at 1 mg/ml). 50 U of the enzyme Hinf I and 11.5 pI of water were added to the mixture, which was incubated for one hour at 37 The digestion mixture was purified on a QIAquick column (QIAGEN), and eluted with 30 p1 of buffer T. 1 p) of this is eluate was loaded on a 1% TBE agarose gel in order to verify that the digestion had been total, and that it had generated 6 restriction fragments, and consequently six libraries of fragments, of 590 bp, 500 bp, 472 bp, 438 bp, 298 bp and 274 bp. The concentration of this DNA was approximately 250 ng/pl according to its absorbance at 260 nm.

V RLR (Recombinating Ligation Reaction).

The RLR reaction (Recombining Ligation Reaction) was carried out 'y incubating determined quantities of restriction fragments Hinf I Bsa I from the genes of ten mutants with the complete template the wild type ponB gene), in the presence of a thermostable DNA ligase. The table IV below reports the composition of the mixtures for RLR.

Table IV RLRl1 RLR 2 RLR 3 RLR 4 Fragments HinfI-BsaI O.5 d 14 2l 2 jLl 5j 1 template (100 ng/[Ld) AMPLIGASE lox Buffer 2 j d 2 gl 2 pl 2 ld 2 1 AMPLIGASE (25 U14*) 1 gd 1 1 .d 1 141 1 ld- H0qsp 20 qsp 20 qsp 20 qsp 20 qsp PI .LI 141 J.L The negative control is identical to the reaction of RLR4, but does not contain thermostable DNA ligase- Thlese different mixtures were covered with s a drop of mineral oil and -incubated in a Perkin-Elmer 9600 thermocycler in 200 M 1 microtubes according to the following program: 94 CC, 5 min.) (94 0 C, 1 min. 65 0 C, 4 min.) x 35 cycles- 41 of each RLR reaction were then added to a PCR reaction mixture i0 containing 10 141 of polymerization buffer, 10 gi of 2 mM dNTPs, 40 pmol of each oligonucleotide Al and A2, and 5 U of Taq DNA polymerase in a final volume of 100 141. This mixture was incubated In a Perkin-Elmer 9600 thermocycler according to the following program: (94'C, 5 min-) (94QC, sec- 460C, 30 sec. 72'C, 1 min.) x 29 cycles (721C, 2 min.). This PCIR )s reactIon permitted specific amplification of the ligation products formed in the course of the RLR reaction, without amplifying the template, since the oligonucleotides Al and A2 are not able to hybridize with the template as shown in Figure 4.

5 141 of each RLR reaction and 10 1 .d of each of the previous PCR reactions were loaded on a 1 TBE agarose gel. After staining with ethidium bromide, the gel was exposed to ultraviolet light, as shown in Figure The analysis of this gel reveals that only the reaction of RLR4 contains, as the negative control, restriction fragments still visible (tracks 4 and The absence of PCR product for the negative control (track 10) reveals not only that the PCR reaction is specific (no amplification of the complete template), but also that the restriction fragments present in the mixture cannot be substituted for the primers to generate a contaminant PCR product under the chosen conditions. In parallel, the presence of a unique band at about 1o 2500 bp in tracks 6, 7 and 8 demonstrates that an RLR product was able to be amplified by PCR for the RLR1, 2 and 3 reactions. These three RLR reactions therefore permitted the regeneration of one or more of the complete genes starting from six libraries of restriction fragments.

VI ANALYSIS OF THE AMPLIFICATION PRODUCTS OF THE RLR REACTIONS 1) Cloning The PCR amplification products of the RLR 1, 2 and 3 reactions were purified with the Wizard PCR Preps system (PROMEGA) and eluted in 45 Il of buffer T. 6 1 of each purified PCR were incubated 1 hour at 37 °C in a mixture containing 3 pl of restriction buffer C, 3 il of BSA (1 mg/ml), 20 U of the Eco RI enzyme, 10 U of the Nco I enzyme and 15 pl of water.

In parallel, two vectors (pARAPONB and pET26b+) were prepared for the cloning. These vectors were linearized by incubating 3 pg of these plasmids for 2 hours at 37 in a mixture containing 3 il of restriction buffer C, 3 pl of BSA (1 mg/ml), 20 U of the Eco RI enzyme, 10 U of the Nco I enzyme and 19 p l of water.

The linearized vectors as well as the digested PCR were purified on a TBE 1% agarose gel with the QlAquick system (QUIAGEN). Each vector or each digested PCR was eluted in 30 l of buffer T.

The ligation of each PCR digested with each of the vectors was carried out according to the conditions described in table V below, and incubated at 16 oC for 16 hours.

Table V 200 jl of chimiocompetent MC1061 cells were transformed with pl of each ligation by a thermal shock and the cells thus transformed were spread over a selection medium.

No clone was obtained after transformation of ligation controls TLpAR and TLpET, thus indicating that the Nco I Eco RI vectors pARAPONB and pET26bt cannot undergo an intramolecular hgation.

22 2) Screening by PCR A first screening of the clones obtained after transformation of the ligations with the vector pARAPONB was carried out by PCR. 42 colonies, 14 from each ligation LpAR1, LpAR2 and LpAR3, were resuspended individually in a PCR mixture containing 5 pl of polymerization buffer, 40 pmol of each oligonucleotide Al and A2, 5 pl of 2 mM dNTPs and 5U of Taq DNA polymerase in a final volume of 50 pl. A negative control was obtained by adding to the PCR mixture 50 ng of the plasmid pBR322 in place of the colony. These 43 tubes were incubated in a Perkin-Elmer 9600 thermocycler according to the following program: (94 0 C, 5 min.) (94 0 C, 30 sec. 46 0 C, sec. 72 0 C, 1 min.) x 29 cycles (72°C, 2 min.). 5 pl of each of these PCR reactions were then incubated for 1 hour at 37 oC in a mixture containing 2 pCl of restriction buffer A, 2 pd of BSA (1 mg/ml) and 5 U of the restriction enzyme is Pvu II in a final volume of 20 p 1 pLI of each of these digestions were loaded on a TBE 1% agarose gel in parallel with 5 pi of each non-digested PCR (thus avoiding possible confusion of non-specific bands of the PCR with a fragment obtained by -restriction digestion). After migration and staining of this gel with ethidium bromide, the bands resulting from the digestion by the enzyme Pvu II were analyzed in order to determine which fragment(s) of initial mutants was/were associated with the others in order to reconstruct an entire gene. This screening reveals the presence of 27 genes carrying one mutation, 7 genes carrying two mutations and 8 genes no longer carrying any mutation.

3) Screening by plasmidic DNA minipreparation The second screening was effected by carrying out an extraction of the plasmidic DNA from 21 clones resulting from the transformation of the ligations with the vector pET26b+ (7 clones of each ligation). 5 pl of the plasmidic DNA thus obtained for each clone were incubated for 1 hour at 37 oC in a mixture containing 1 pl of restriction buffer C, 6 U of the enzyme Pst I, 3 U of the enzyme Nco I and 6 U of the enzyme Eco RI in a final volume of 4l. 5 pLI of each of these digestions were loaded on a TBE 1% agarose gel.

After migration and staining of this gel with ethidium bromide, the bands resulting from the digestion by the Pst I enzyme were analyzed in order to determine which fragment(s) of the initial mutants had associated with the others in order to reconstruct an entire gene. This screening reveals the presence of 13 genes carrying a mutation, 5 genes carrying two mutations and 3 genes no longer carrying a mutation.

4) Statistical Analysis of the recombinations.

According to the position of each mutation as compared to the cutting sites of the enzymes Hinf I and Bsa I, as represented in Figure 6, it is possible to calculate the probability of obtaining in the course of the RLR reaction the creation of a gene carrying 0, 1, 2, 3, or 4 of the mutations of the initial genes.

Thus, by considering that the RLR reaction is totally random the probabilities P are as follows: P(0 mutation) 30.24% 20 2 0 n P(1 mutation)= 1 n (i 44.04% 4[ 1 -O-a 10 i P(2 muTraions) IL O-a 1Y-. P)U J =21-44% -I a n 1 0 P(3 mutations)= i[(1 U (i ]4.04% P(4 muiaions) 0.24% The two screenings carried out give results close to these statistical predictions, as reported in table VI below, thus indicating that the RLR reaction is quasi-random. A slightly higher proportion of genes carrying one mutation, to the detriment of the genes carrying zero mutation, is observed.

This phenomenon could be attributed to a weak toxicity of the ponB gene already observed and to the slight of expression leakage of vectors pARAPONB and pET26b'-. which would favor the selection of genes carrying an inactivating mutation.

Table VII 0 mutation 1 mutation 2 mutations 3 mutations 4 mutations Statistics 30.24 44.04 21-44 4.04 0.24 PCIR 21 63 16 0 0 Screening MIni- 14 62 24 0 0 preparation Screening BIOGRAPHICAL REFERENCES 1) Broome-Smith J.K, Edelman Al, Yousif S. and Spratt B.G., (1986), The nucleotide sequence of the ponA and ponB genes encoding penicillin-binding proteins 1A and 1 B of Eschenichia coi K12, Eur. J. Biochem., 147, 437-446.

2) Caldwell R.C. and Joyce 1992, Randomization of genes by PCR mutagenesis, PCR Methods and Application, 2, 28-33.

3) Cagnon Valverde V. and Masson (1991), A new family of sugar inducible expression vectors for Eschenichia coli, Prot.

Eng., 4, 843-847- 4) Hanahan (1985), Techniques for transformation of coi, in DNA cloning: a practical approach, Glover D.M. IRL Press, Oxford vol 1, 109-1 Maniatis Fristch E.F. and Sambrook (1982), Molecular cloning. A laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 6) Landt et al., Gene, 96, 125-128, 1990- 7) Lef~vre Topological Analysis of the Penicillin Binding Protein 1 b of Escherichia col, 19 97, These_ 8) Lef~vre Rdmy M H. and Masson 1997 Alaninestretch scanning mutagenesis: a simple and efficient method to probe protein structure and function, Nuc. Acids Res., 25, 447- 448.

9) Lef~vre F, R~smy M.H_ and Masson JM., 1997 Topographical and functional investigation of Esctichia coi Penicillin-Binding Protein lb by alanine stretch scanning mutageflesis, J. Bacteriol., 179, 4761-4767.

Lyamichev Mast Prudent Kaiser Takova T., Kwiatkowski Sander de Arruda Arco Neri B.P. and Brown 1999, Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes, Nature Biotechn ology, 17, 292-296.

11) Picard et al., Nuc. Acids Res., 22, 2587-2591, 1994.

12) Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature, 370, 14 1-144.

13) Studier F.W and Moffatt 1986, Use of bacteriophage 17 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol, 189, 113-130- 14) Zhao, Giver L. Shao Affholter J.A_ and Arnold 1998, Molecular evolution by staggered -extensionl process (StEP) in vitro recombinationi, Nature Biotech.. 16, 258-261.

Claims

1. A process of obtaining recombinant polynucleotide sequences in vitro starting from a library of polynucleotide sequences, which includes the following steps: a) preparing a library of double-stranded polynucleotide sequences from a native gene by steps of: i) successive directed mutagenesis; ii) error prone PCR; iii) random chemical mutagenisis; iv) in vivo random mutagenisis; or v) combining genes of close or distinct families within the same or different species in such a way as to arrange a variety of polynucleotide sequences in an initial library; b) generating fragments from the library; c) denaturation of the fragments; d) hybridization of said fragments with one or more assembling templates; e) ligation of said fragments in order to obtain Srecombined polynucleotide sequences; and f) selection of recombined polynucleotide sequences which have advantageous properties as compared to the corresponding properties of one or more reference sequences.

2. A process according to claim 1, wherein the fragments are denatured in the presence of one or more assembling templates.

3. A process according to claim 1 or claim 2, which 35 includes, after step and before step repeating steps and with the ligated and non-ligated fragments resulting from step H:\RBe11\Keep\51717-99-doc 31/10/03 28

4. A process according to any one of claims 1 to 3, which includes separation of the recombined polynucleotide sequences from the assembling template or templates before step A process according to any one of claims 1 to 4, which includes amplification of the double-stranded recombined polynucleotide sequences before step

6. A process according to any one of claims 1 to which includes a further step before step of cloning of the recombined polynucleotide sequences.

7. A process of claim 6, in which the recombined polynucleotide sequences are cloned after separation of the recombined strands of the template or templates and obtaining the corresponding double strand.

8. A process according to any one of claims 1 to 7, in which the ends of the fragments generated at step (a) are such that there can be adjacent hybridization of these ends on the assembling template or templates at step (c) Sand ligation of these fragments with each other at step 25

9. A process according to any one of claims 1 to 8, in which the polynucleotide sequences of the initial library have zones of homology either between themselves or with the assembling templates, so as to generate at step ends of fragments which permit the adjacent hybridization of these ends on the assembling template or templates at step and ligation of these fragments with each other at step A process according to any one of claims 1 to 9, in which steps and are carried out simultaneously. H;\RBell\Keep\51717-99.doc 31/10/03 29

11. A process according to any one of claims 1 to in which the generation of fragments from the polynucleotide sequences at step is carried out in a controlled or a random manner.

12. A process according to any one of claims 1 to 11, in which step consists of subjecting the polynucleotide sequences of the initial library to hydrolysis by the action of one or several restriction enzymes.

13. A process according to any one of claims 1 to 12, in which the degree of recombination and the position of the points of recombination of the recombined polynucleotide sequences are determined by the fragmentation described in step which is carried out in a controlled manner.

14. A process according to any one of claims 9 to 13, in which the random generation of fragments from the polynucleotide sequences at step is carried out by any Sknown enzymatic or mechanical means. 25 15. A process according to any one of claims 1 to 14, in which enzymes capable of recognizing and of cleaving the non-hybridized ends of the fragments in a specific manner are added at step and/or step when said ends overlap with other hybridized fragments on the same template.

16. A process according to claim 15, in which the enzyme flap endonuclease is added at step and/or step (d)

17. A process according to any of claims 1 to 16, in which a ligase which is active at high temperature is used H:\RBell\Keep\51717-99.doc 31/10/03 30 at step

18. A process of claim 17, wherein the ligase is thermostable.

19. A process according to any one of claims 15 to 18, in which the enzymes capable of specifically recognizing and of cleaving the non-hybridizing ends of the fragments added at step and/or at step have the same thermoresistance and high temperature activity properties as the ligase used at step A process according to any one of claims 1 to 19, in which the initial library of double-stranded polynucleotide sequences further comprises synthetic sequences which are fragmented at step or which can form the fragments of step

21. A process according to any one of claims 1 to 13 and 15 to 20, in which step consists of subjecting the initial library to hydrolysis by the action of restriction enzymes from which a large number of cleavage sites are present in the polynucleotide sequences of the initial library, or by combining several restriction enzymes.

22. A process according to any one of claims 1 to 11 and 14 to 20, in which step consists of a random treatment with the DNAse I of an initial library of partially heterologous, double-stranded polynucleotide sequences.

23. A process according to any one of claims 14 to 18 or 20 to 22, in which fragments generated by a random treatment are used as templates for each other, for the 35 hybridization during step or the RLR reaction of steps and simultaneously. ooo. H:\RBell\Keep\51717-99.doc 31/10/03 31

24. A process according to any one of claims 12 to 23, in which step is carried out by combining at least two distinct libraries of fragments generated separately at step obtained from the same initial library by treatment with different restriction enzymes. A process according to claim 24, in which the fragments obtained at step by treatment with restriction enzymes are used as templates for each other, for the hybridization during step or of the RLR reaction steps and simultaneously.

26. A process according to any one of claims 1 to in which the fragments of step are obtained by amplification reactions directed by the polynucleotide sequence of the initial library.

27. A process according to claim 26, in which the amplification reactions are carried out with oligonucleotide primers permitting generation of fragments whose ends are adjacent along the whole length of the assembling sequence.

28. A process according to claim 26, in which the amplification reactions are carried out with oligonucleotide primers permitting generation of fragments having common sequences, said fragments serving as an assembling template for each other at step or at step

29. A process according to any one of claims 1 to 28, in which at step the initial library is fragmented into n fragments, n being greater than or equal to 3. 35 30. A process according to any one of claims 1 to 29, in which the assembling template at step or step (c) is a polynucleotide sequence resulting from the initial H:\RBell\Keep\51717-99.doc 31/10/03 32 library or a single or double-stranded consensus sequence of said library.

31. A process according to any one of claims 1 to in which single or double-stranded oligonucleotides complementary only at the 3' end of one fragment and at the 5' end of the adjacent fragment are used as assembling template at steps or

32. A process according to any one of claims 1 to 31, in which single or double-stranded oligonucleotides of variable length are added at step or in addition to the template.

33. A process according to any one of claims 1 to 32, in which before step the recombined polynucleotide sequences are separated from the assembling template by means of a label present on the assembling template or on the recombined polynucleotide sequences.

34. A process according to any one of claims 1 to 33, in which the recombinant polynucleotide sequences obtained at step and optionally cloned are screened in order to select the recombined polynucleotide sequences or the 25 clones having advantageous properties as compared to the corresponding properties of the reference sequences. A process according to any one of claims 1 to 34, in which the initial library of polynucleotide sequences is formed by one or several limited libraries prepared by a process according to any one of claims 1 to 34, .•optionally mixed with other polynucleotide sequences.

36. A recombinant polynucleotide sequence having one 35 or several advantageous properties when compared to the *corresponding properties of reference sequences, obtained and selected by a process according to any one of claims 1 H:\RBell\Keep\51717-99.doc 31/10/03 33 to

37. A vector comprising a polynucleotide sequence according to claim 36.

38. A cellular host transformed by a recombined polynucleotide sequence according to claim 36 or by a vector according to claim 37. S *5 S S SSS S

39. sequence

41. 25 35 A protein encoded by a recombined polynucleotide according to claim 36. A library formed from a) recombinant polynucleotide sequences according to claim 33, b) vectors according to claim 34, c) host cells according to claim 35, or d) of proteins according to claim 36. A library of fragments wherein: a) said fragments come from at least two libraries of polynucleotide sequences generated from a native gene by the steps of: i) successive directed mutagenesis; ii) error prone PCR; iii) random chemical mutagenesis; iv) in vivo mutagenesis; or v) combining genes of close or distinct families within the same or different species in such a way as to arrange a variety of polynucleotide sequences in an initial library according to step a) of claim 1; b) some of said fragments are substantially homologous to an assembling template, on which they are capable of hybridising; and a) when the ends of two hybridized fragments are S 5 S S. *S S SaSS H:\RBell\Keep\51717-99.doc 31/10/03 34 adjacent, they are capable of being ligated by a ligase.

42. Use of a library according to claim 38 in a process of obtaining in vitro recombinant polypeptide sequences using a ligase.

43. A process according to claim 1, of obtaining in vitro recombined polynucleotide sequences, substantially as herein described with reference to any of the figures or the example.

44. A library of fragments according to claim 38, substantially as herein described with reference to any of the figures or the example. Dated this 3 1 st day of October 2003, PROTEUS By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia 0S S to H:\RBell\Keep\51717-99.doc 31/10/03