Patent Application: US-46839104-A

Abstract:
the presented invention refers to a method for the creation of gene libraries wherein a defined number of adjacent nucleotides is exchanged and gene libraries are produced which code for protein variants having more manifold amino acid exchanges and a more homogenous distribution of mutations than can be obtained using conventional methods . dna - strands are incorporated at random positions into a gene of interest . then parts of the donor strands and parts of the gene sequence that is flanking these strands are removed , however , a defined number of nucleotides that originate from the donor strand remain in the gene at the place of a defined number of nucleotides of the original gene having been removed from it . combined with a selection step after the incorporation of the donor strand into the gene it can be ensured that the nucleotides to be exchanged / introduced are in a specific reading frame . when the nucleotides of the donor strand that remain in the genes are degenerate , gene libraries can be produced with variants that have any codon at any position .

Description:
the invention consists of ( also refer to fig1 ): a .) the insertion of a piece of dna into a gene at different , randomly positioned sites . b .) the directed removal of this piece of dna and of a defined number of adjacent nucleotides of said gene , while instead at this position a defined number of nucleotides from said inserted piece of dna is remaining . in detail the invention is marked by the following steps ( also refer to fig2 ): 1 .) into molecules of a gene or of a dna sequence , preferably as part of a vector or any other circular form , exactly one double strand breakage per molecule is performed by mans of molecular biological methods ( fig2 , i ). this can , for example , be achieved by treating the dna with a ) an enzyme that site - nonspecifically introduces single strand breaks ( e . g . dnase i ) and a single strand specific nuclease ( e . g . s1 nuclease ), b ) an enzyme that site - specifically introduces single strand breaks , a 5 ′- 3 ′ exonuclease , dna - polymerase and single strand specific nuclease ( e . g . s1 nuclease ), c ) an enzyme that site - nonspecifically introduces double strand breaks ( e . g . modified variants of restriction enzymes that have lost their sequence specificity ), or d ) a transposon and a transposase that does not have a sequence specificity . it is preferred that an ensemble of gene variants is produced in which the double strand breaks are located at different positions and ( apart from case d ) in which the double strand breaks lead to blunt dna ends . in case d ) the double strand breakage is achieved under simultaneous incorporation of a dna strand ( transposon ) and possibly the doubling of several nucleotides from the gene . 2 .) into the gene variants of this ensemble a dna strand ( donor - strand ) is incorporated by blunt - end - ligation ( fig2 , ii ). in the case of the utilization of a transposase ( 1d ) the donor - strand , as a transposon , has been incorporated already during the previous step . it is preferred that the donor - strand encodes a genetically selectable marker , e . g . a resistance against an antibiotic . it is further preferred that the expression of this marker is dependent on that the inserted donor - strand is incorporated into the correct reading frame of the gene . preferably , the donor - strand contains recognition sites for restriction enzymes of type iis . such obtained dna constructs can be completely or partially amplified by pcr 30 , 31 and / or can be amplified in and isolated from microorganisms after having transformed them . it is preferred that the growth of the microorganisms is performed in culture media containing antibiotics against which the microorganisms are resistant due to the gene product that is encoded on the donor - strand . 3 .) by restriction digestion with said restriction enzymes of type iis the donor - strands are mostly removed from the amplified gene variants . the dna ends are made blunt by treatment with a dna polymerase or with a single strand specific nuclease ( fig2 , iii ). it is preferred that the positions of the recognition sites of said restriction enzymes of type iis are chosen such that in addition to the removal of most of the donor - strand a defined number of n nucleotides is removed from the original gene and that at their position a defined number of m nucleotides from the original donor - strand is remaining . it is preferred that these remaining m nucleotides are degenerate , meaning that in the gene variants of the ensemble different nucleotide compositions are remaining . it is preferred that exactly three nucleotides are replaced ( n = 3 , m = 3 ). in case that the variability of the nucleotide composition in the close proximity ( 10 to 40 base pairs ) of the ends of the donor - strand is restricted , e . g . by conserved sequences of a transposon , it is preferred that the donor - strand contains several recognition sites for restriction enzymes of type iis . these are preferably positioned in a way that the donor - strand is removed from the gene bit by bit in several cycles of a ) restriction digestion with one or two of said enzymes , b ) when necessary , treatment to create blunt dna ends and c ) followed by the fusion of the dna ends by intramolecular ligation , until the entire donor - strand apart from m nucleotides is removed together with a defined number n nucleotides from the original gene ( fig2 , iv and v ). these remaining m nucleotides preferably are degenerate , it is preferred that exactly 3 nucleotides are replaced ( n = 3 , m = 3 ). 4 .) by intramolecular blunt - end - ligation the dna - ends of the variants of the ensembles are closed and complete , continuous genes are obtained ( fig2 , vi ). the genes can be subjected to a further round of introduction of mutations . alternatively the genes can be expressed in vivo after transformation of an expression host or in vitro by using an in vitro — translation system to yield the protein variants that are encoded by the genes . the here disclosed method is so far the only method for mutagenesis that allows the random exchange of several adjacent nucleotides in a gene . so far several methods of molecular biology have been published that are based on random double strand breaks of dna strands or on the insertion of dna sequences , including transposons at random positions into dna strands . these methods , however , are limited to the experiments to find new termini for proteins 32 , to randomly delete parts of protein sequences or insert additional sequences into proteins 33 . they have not been applied and , by themselves , they are not even applicable to exchange nucleotides at random positions in dna sequences in such a way that single amino acids in the accordingly encoded proteins are exchanged to produce a repertoire of genes whose products are distinguished to each other in the type of a defined number of amino acids . it is predicted that the fraction of any theoretically possible mixture of nucleotides within the degenerate part of the donor dna can be accurately adjusted , e . g . in such a way that in an area of 3 degenerate nucleotides each amino acid is represented by exactly one codon . it is predicted that the disclosed method allows incorporating mutations not only into the entire lengths of a gene but also into limited parts of genes . for example , after incorporation of the donor - strands these parts can be amplified by pcr using flanking primers , the amplified products can be fused into the complete gene by the use of gensoeing 34 and the such modified gene can then be further subjected to the described protocol . it is further predicted that during the stepwise removal of the donor - strands , required restriction sites of type iis are only created in the process , e . g . by restriction and religation . it is predicted that donor - strands that are incorporated into genes as transposons by the action of a transposase can be modified with mutations within the transposase recognition sequence that necessary for the transposition , such that new recognition sites for restriction enzymes of type iis are created within the transposase recognition sequence . it is predicted that there are other techniques that can be applied to introduce double strand breaks into dna than the ones exemplary indicated in the description of the invention . it is predicted that by applying immobilization techniques genes with incorporated donor - strands can be physically separated from genes that do not contain donor - strands or that by applying immobilization techniques donor - strands that are incorporated into genes can be physically separated from donor - strands not incorporated into genes . the possibilities and the approach of the invention will become even clearer in the following example . the example of practicing the invention is understood to be exemplary only , and do not limit the scope of the invention or the appended claims . a person of ordinary skill in the art will appreciate that the invention can be practiced in many forms according to the claims and disclosure here . introduction of codon mutations into the gene of the green fluorescent protein ( gfp ) ( also refer to fig3 and 4 ) 1 .) introduction of randomly positioned double strand breaks in the plasmid pgfp the plasmid pgfp ( clontech , palo alto , usa ) contains the gfp - gene under the control of the lac - promoter . for the amplification in e . coli the plasmid contains the gene for the resistance against ampicillin . e . coli xl1 - blue cells were transformed with pgfp and from 200 ml of a culture of the transformed cells 300 μg pgfp dna were prepared ( maxikit , quiagen , hilden , germany ). in 200 μl 33 mm tris / hcl , ph 7 , 5 , 10 mm mgcl 2 and 50 μg / ml bsa 40 μg pgfp were incubated with 0 . 01 mu dnase i ( roche diagnostik , penzberg , germany ) for 5 min at 28 ° c . the reaction was stopped by addition of 20 mm edta ( final conc .) and cooling on ice . the analysis of the reaction by agarose gel electrophoresis revealed that approx . 40 % of pgfp had been converted into the open - circular form . this open circular dna was isolated using preparative agarose gel electrophoresis . in 100 μl 7 . 4 × s1 buffer ( mbi fermentas , st . leon roth , germany ) 5 μg of the open - circular form were incubated with 100 u s1 nuclease ( 1 μl , mbi fermentas , st . leon roth , germany ) for 2 h at 16 ° c . after which the reaction was stopped with 10 μl s1 - stop solution . the analysis of the dna by agarose gel electrophoresis revealed that approx . 50 % of the open circular dna was linearised . this linearised dna was isolated by preparative agarose gel electrophoresis . the gene of chloramphenicol acetyltransferase ( cat ) was amplified by pcr with the primers nns ggg cct ggg tct cct cct ggc gag aaa aaa atc act gga tat acc ( seq . id no : 1 ) and ggc gta gct cct cgc gtt taa ggg ( seq . id no : 2 ) and the plasmid pacyc184 ( neb , beverly , mass ., usa ) as template . the pcr was performed following standard protocols ( neb , beverly , mass ., usa ), 30 cycles were performed applying an annealing temperature of 55 ° c . and an extension time of 45 sec . vent - polymerase was used ( neb , beverly , mass ., usa ). the pcr product was precipitated with etoh and resuspended in a small volume te to give a concentration of 150 ng / μl . in 50 μl ligase buffer ( gibco brl , eggenstein , germany ) ( final volume ) 10 μl linearised plasmid ( approx . 300 ng , refer to 1 .) and 14 μl pcr product ( approx . 2 μg , refer to 2 .) were incubated with 5 u t4 - dna ligase ( gibco brl , eggenstein ) for 20 h at 16 ° c . subsequently the ligation mix was desalted by microdialysis and used to transform xl1 - blue cells by electroporation . transformed cells were plated on dyt - agar including 100 μg / ml ampicillin , 8 μg / ml chloramphenicol and 1 mm iptg . growth of transformed bacteria was basically limited to cells transformed with plasmids that contained the pcr fragment under the control of the lac - promoter in the correct reading frame after a start codon . approx . 10000 transformants were obtained . 95 colonies were analyzed by colony - pcr using the primers cca tga tta cgc caa gct tgc ( seq . id no : 3 ) ( binds to the 5 ′- end of the gfp - gene and gtg ctt att ttt ctt tac ggt c ( seq . id no : 4 ) ( binds within cat - gene ) for whether an insertion of the pcr - product into the plasmid had occurred within or outside the gfp gene and whether the insertion into the gfp gene had occurred in the correct direction of translation and at positions randomly distributed . from approx . 80 % of the transformants a fragment between ca . 270 to ca . 1000 bp in length could be amplified ( see fig4 a as an example . for all those variants the insertion of the pcr - product had occurred within the gene sequence of gfp in a way that the direction of translation of the gene for chloramphenicol acetyltransferase lies in the same as for the gene for gfp . all colonies of the transformed bacteria were collected from the agar plates , pooled and plasmid dna was prepared ( mini kit , quiagen , hilden , germany ). 2 μg of the plasmid dna , which represents a repertoire of pgfp with randomly inserted pcr products , was completely digested with bseri ( neb , beverly , mass ., usa ). this restriction enzyme of type iis cuts outside its recognition site ctcctc ( fig3 , iii ). the products of the restriction digestion were treated with klenow fragment , subsequently separated by agarose gel electrophoresis and the dna band that had a length of approx . 3 . 4 kb was isolated from the agarose ( qiaexii , qiagen , hilden , germany ). ca . 40 ng of the dna were incubated in 50 μl ligation buffer with 1 u t4 - 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