Patent Application: US-51132705-A

Abstract:
the present invention describes an in vitro transposition - based methodology for generation of deletion derivatives of polypeptides . an artificial transposon containing at least partly within its transposon ends a modification with translation stop codons in three reading frames is provided . in the method , transposition complexes are assembled using the modified transposon and essentially random integrations into the target plasmid , containing a polypeptide coding nucleic acid of interest , are recovered as a plasmid pool . subsequent manipulation steps including restriction enzyme digestions and ligation result in pools of mutant clones from which deletion derivatives of a polypeptide coding nucleic acid of interest and its respective deletion polypeptides could be produced .

Description:
it has been published previously that protein engineering applications will benefit from mu - based transposon strategies since it was established that any dna sandwiched between mu ends could be utilised as artificial transposons ( haapa et al . 1999a ). in , principle insertion mutations ( e . g . by addition of epitope tags or protein domains ) and deletion mutations ( by addition of translation stop codons ) were foreseen with this strategy . however , introduction of a translation stop codon between transposon ends would leave a number of encoded amino acid residues into the protein &# 39 ; s c - terminus . given that an effective mu end is about 50 bp in length , minimally this strategy would leave approximately 18 extra amino acids attached in the protein c - terminus . extra amino acids may interfere with the protein function , therefore it would be better to add the stop codons as close as possible to the transposon end . by modifying the nucleotides of the mu r - end ( total of 7 nucleotides were changed , 5 of said nucleotides reside in mu r1 sequence ), we managed to place three stop codons in three reading frames very close to the mu r - end resulting in transposons that still surprisingly retained their ability to form transposition complexes that were competent for transposition chemistry , i . e . they facilitated the integration of the transposon in ? vitro into a target plasmid . in essence , all the possible c - terminal deletion variants can be generated . we designed an artificial cat - mu ( stop )- transposon ( seq id no : 2 ) conferring resistance to chloramphenicol and tn7 - kan ( stop )- transposon ( seq id no : 7 ) conferring resistance to kanamycin . both contained in their ends modified base pairs providing three stop codons in three reading frames ( fig1 and 2 ). the gene mediating resistance to chloramphenicol is used as a selectable marker . the term “ selectable marker ” refers to a gene that , when carried by a transposon , alters the ability of a cell harboring the transposon to grow or survive in a given growth enviroment relative to a similar cell lacking the selectable marker . the transposon nucleic acid of the invention preferably contains a positive selectable marker . a positive selectable marker , such as an antibiotic resistance , encodes a product that enables the host to grow and survive in the presence of an agent , which otherwise would inhibit the growth of the organism or kill it . the transposon nucleic acid of the invention may also contain a reporter gene , which can be any gene encoding a product whose expression is detectable and / or quantitatable by immunological , chemical , biochemical , biological or mechanical assays . a reporter gene product may , for example , have one of the following attributes : fluorescence ( e . g ., green fluorescent protein ), enzymatic activity ( e . g ., luciferase , lacz / β - galactosidase ), toxicity ( e . g ., ricin ) or an ability to be specifically bound by a second molecule ( e . g ., biotin ). the use of markers and reporter genes in prokaryotic and eukaryotic cells is well - known in the art . in a preferred embodiment the transposon nucleic acid of the invention may also contain genetically engineered restriction enzyme sites . for example , the selectable marker gene within the transposon of the invention may influence the protein expression when a construct obtained by the method of the invention is inserted into a protein expression plasmid . it is therefore desirable to engineer a pair of unique restriction sites to flank the selectable marker gene . the marker can then be removed easily by the use of these sites and thus the final expression construct would not contain the marker gene . hence , one embodiment of the invention provides a transposon nucleic acid comprising a genetically engineered translation stop signal in three reading frames at least partly within a transposon end sequence , or preferably within transposon end binding sequence , recognised by a transposase ( i . e . at least one conserved nucleotide of the end sequence has been modified , preferably two , three , four or more conserved nucleotides have been modified ). preferably , the transposon nucleic acid of the invention comprises mu or tn7 transposon sequence . more preferably the transposon nucleic acid of the invention comprises mu r - end sequence , e . g ., the sequence of seq id no : 1 or seq id no : 5 ( mu - r end sequence not including 5 ′ overhang , which thus can vary ). in a transposon end sequence of the transposon nucleic acid of the invention , translation stop signals of three reading frames are in 5 ′- to - 3 ′ direction , preferably in succession close to each other at a very end of a transposon , thus the three stop signals are as near as possible the flanking sequence after the transposon is incorporated into a target . furthermore , the transposon end sequences , which participate in the assembly of the transpososome discussed above , can be different from each other or they can be in different nucleic acid molecules . preferably , both transposon end sequences participating in the transpososome have similar sequences ( i . e . they are located as inverted terminal repeats ). the transposon nucleic acid of the invention is exemplified here by transposons of mu ( examples 1 - 3 ) or tn7 ( example 4 ) system . however , a person skilled in the art understands that teachings of this invention can be utilised in other transposon systems as well . another embodiment of the invention is a method for producing a deletion derivative of a polypeptide coding nucleic acid comprising the steps of : ( a ) performing a transposition reaction in the presence of a target nucleic acid containing a polypeptide coding nucleic acid ( e . g . a gene ) of interest and in the presence of a transposon containing a genetically engineered translation stop signal sequence in three reading frames at least partly within a transposon end sequence recognised by a transposase , ( b ) recovering a target nucleic acid having said transposon incorporated in said gene . the transposition reaction ( a ) includes a transposon in a form of linear dna molecule , transposase protein ( e . g . mua ), and a target dna as macromolecular components . additionally , the transposition reaction contains suitable buffer components including mg 2 + ions critical for chemical catalysis . buffer components such as glycerol and dmso ( or related chemicals or solvents ) somewhat relax the requirements for transposition reaction ( savilahti et al . 1995 ). transposon dna , in principle , can be of any length given that it in each end contain a transposon ( e . g . mu or tn7 ) end sequence . typically , target dna is in a form of circular plasmid . however , any double - stranded dna molecule more than 25 bp is expected to serve as efficient target molecule ( savilahti et al . 1995 , haapa - paananen et al . 2002 ). in transposition reaction the reaction components are incubated together ; during the incubation transposition complexes first form and then react with target dna splicing the transposon dna into target dna . this process yields transposon integrations into target molecules . the stoichiometry of the reaction ( excess target ) generates target molecules each with a single integrated transposon . most importantly , the integration site in each molecule can be different . even though some sites in dna are somewhat more preferred than others most of the phosphodiester bonds in dna will be targeted ( haapa et al . 1999ab , haapa - paananen et al . 2002 ). in practice this means that the integration sites are selected essentially randomly . in the examples below deletion mutant libraries were planned to cover the gene of interest at least 10 - fold , i . e . when the target gene was approximately 600 bp , the final pool should contain of a minimum of 6000 mutants . as a test protein we utilised 23 kda yeast mso 1 protein ( aalto et al . 1997 ). those skilled in the art can easily design different strategies for mutant library construction as such strategies are well - known in the art ( see , e . g ., sambrook et al . 1989 , sambrook and russell 2001 ). a mutant library was produced as described in example 2 . target nucleic acids with a transposon insertion were isolated by size - selective preparative agarose gel electrophoresis . a person skilled in the art may design different isolation methods as such methods are well - known in the art ( see , for example , current protocols in molecular biology , eds . ausubel et al , john wiley & amp ; sons : 1992 ). we screened individual deletion mutants by restriction analysis ( fig3 ). this analysis demonstrates that in the library , there are variants of different sizes . a person skilled in the art can easily utilise different screening techniques . the screening step can be performed , e . g ., by methods involving sequence analysis , nucleic acid hybridisation , primer extension or antibody binding . these methods are well - known in the art ( see , for example , current protocols in molecular biology , eds . ausubel et al , john wiley & amp ; sons : 1992 ). we sequenced 23 c - terminal mutants derived from example 2 . all the mutants carried the translation stop codons in three reading frames . finally , the protein expression analysis ( fig4 ) demonstrated that different deletion variant proteins are produced . probably due to lack of resolution in the utilised gel system , the supposedly expressed protein was not detectable when the deletion derivative was 8 kda or smaller . alternatively , very small versions of the mso1 protein may be proteolytically degraded inside the cells . a further embodiment of the invention is a kit providing means for producing deletion derivatives of protein coding nuclear acid sequences . the kit comprises the transposon nucleic acid of the invention . the kit can be packaged in a suitable container and preferably it contains instructions for using the kit . the results of the invention show that , unexpectedly , it is possible to substantially modify conserved sequences of transposon ends without critically compromising the competence of the modified transposon to assemble transposition complexes and thereafter carry out transposition chemistry . thus , the invention provides a straightforward solution to the problem of extra amino acids attached in the protein c - terminus of the deletion derivative which could be produced by a conventional transposition system , wherein the transposon used contains the translation stop signals between the transposon ends . the present invention is further described in the following examples , which are not intended to limit the scope of the invention . in vitro transposition reaction ( 25 μl ) contained 720 ng cat - mu ( stop ) transposon as a donor , 500 ng plasmid phis6 - mso1 as a target nucleic acid , 0 . 2 μg mua , 25 mm tris - hcl at ph 8 . 0 , 100 μg / ml bsa , 15 % ( w / v ) glycerol , 0 . 05 % ( w / v ) triton x - 100 , 126 mm nacl and 10 mm mgcl 2 . the reaction was carried out at 30 ° c . for 4 h . further details and variables of in vitro mu transposition are described in haapa et al . 1999ab and savilahti et al . 1995 , incorporated herein by reference . in vitro transposition reactions with stop - mu were performed essentially as described in haapa et al . ( 1999a ) with the exception that they contained 720 ng donor dna ( stop - mu × 3 ) and 0 , 88 μg mua . ten reactions were pooled , phenol and chlorophorm extracted , ethanol precipitated , and resuspended in 30 μl of water . several 1 μl aliquots were electrotransformed , each into 25 μl of dh5α electrocompetent cells , as described ( haapa et al . 1999a ). transposon - containing plasmid clones were selected on lb plates containing ap and cm . a total of ˜ 6 × 10 5 colonies were pooled and grown in selective lb - ap - cm medium at 37 ° c . for 3 h after which plasmid dna was prepared from the pool with qiagen plasmid midi kit . this plasmid preparation was subjected to a xhoi - hindiii double digestion and preparative agarose gel electrophoresis . the dna fragment corresponding to transposon insertions into the mso1 - containing dna fragment was isolated with qiaquick gel extraction kit ( qiagen ). this fragment was then ligated into the plasmid ph is 6 - mso1 vector xhoi - hindiii backbone to generate a construct pool with transposon insertions located only within the mso1 gene . after ligation , a pool of plasmids from ˜ 5 × 10 4 colonies was prepared as described above . approximately 110 000 colonies were pooled . transposon - carrying mso1 fragments were cloned into clean vector backbone as described above and approximately 11 000 colonies were pooled in the final c - terminal deletion mutant library . at all stages , the transformants were selected with ap and cm . mutant clones were analyzed for deletions by bamhi digestion and dna sequencing . for protein expression analysis , single mutant plasmids were introduced into bl21 ( de3 ) expression strain . selective medium was inoculated with o / n culture of bacteria containing mutant plasmid and grown until od 600 was 0 . 4 - 0 . 7 . protein expression was induced with 1 mm iptg for 3 hours and samples were withdrawn for sds - page analysis . bacterial lysates were run on 15 % gels and stained with gelcode blue stain ( pierce ) as recommended by the supplier . in vitro tn7 transposition reaction ( 20 μl ) contained 40 ng tn7 - kan ( stop ) transposon ( seq id no : 7 ) as a donor , 100 ng plasmid puc19 as a target nucleic acid , 7 ng tnsa protein , 10 ng tnsb protein , 20 ng tnsc * protein , 25 mm tris - hcl at ph 8 . 0 , 50 μg / ml bsa , 2 mm dtt and 2 mm atp . the reaction mixture was pre - incubated at 37 ° c . for 10 min before addition of 30 mm magnesium acetate . after the addition the reaction was carried out at 37 ° c . for 1 h . the reaction mixture was precipitated with n - butanol to reduce the ionic strength and to concentrate dna prior to electroporation ( thomas , 1994 ) and resuspended in 10 μl of water . 5 μl aliquot was electrotransformed into 50 μl of dh10b ( epicentre technologies ) electrocompetent cells . transposon - containing plasmid clones were selected on lb plates containing kanamycin ( 20 μg / ml ). approximately 20000 kanamycin resistant colonies were recovered per 1 μg target dna . three clones were picked from the transformation plates and grown in lb - kn medium at 37 ° c . overnight after which plasmid dna was prepared from the cultures with qiaprep spin miniprep kit . the tn7 - kan ( stop ) transposon insertion sites were analyzed by dna sequencing . all the mutants carried the translation stop codons in six reading frames and in each case , the integrated transposon was flanked by a 5 - bp target site duplication generated in tnsabc *- mediated transposition . bacterial cultures were grown in luria broth supplemented with appropriate antibiotics : ampicillin ( ap ) at 100 μg / ml , chloramphenicol ( cm ) at 10 μg / ml and kanamycin ( kn ) at 20 μg / ml when required . escherichia coli strains were dh5α ( life technologies ), bl21 ( de3 ) ( novagen ), and dh10b ( epicentre technologies ). mua protein was purified in collaboration with finnzymes ( espoo , finland ) essentially as described ( baker et al . 1993 , haapa et al . 1999a ). tnsa , tnsb and tnsc * proteins were purchased from new england biolabs . restriction enzymes and t4 dna ligase were from new england biolabs and promega , triton x - 100 from roche . standard dna techniques were performed as described ( sambrook and russell 2001 ). enzymes were used as recommended by suppliers . sequencing was carried out at the sequencing service unit of the institute of biotechnology , university of helsinki . plasmid phis6 - mso1 contains the 633 bp mso1 gene as an insert ( aalto et al . 1997 ). the cat - mu ( stop ) transposon ( 1254 bp ) is a derivative of the cat - mu transposon ( haapa et al . 1999a ), and they encode resistance to chloramphenicol ( fig1 and 2 ). the cat - mu ( stop )- transposon ends were engineered to carry translation stop signals for both 5 ′- to - 3 ′ directions of dsdna in all three reading frames . the tn7 - kan ( stop ) transposon is a derivative of the pgps1 . 1 transposon ( new england biolabs ) and it encodes resistance to kanamycin . the tn7 - kan ( stop ) transposon ends were engineered to carry translation stop signals for both 5 ′- to - 3 ′ directions of dsdna in all three reading frames . tn7 - kan ( stop ) transposon sequence is 4814 bp in length ( seq id no : 7 ) and nucleotides 3093 - 4791 set forth in seq id no : 7 constitutes the transposable element . modified nucleotides were at the positions of 3095 , 3097 , 3099 , 3101 , 3103 , 4781 , 4783 , 4785 , 4787 , and 4789 set forth in seq id no : 7 . tn7 - kan ( stop ) transposon was constructed from pcr - amplified fragments . the transposable fragment was amplified with primers 5 ′ acg gtg agt gag tag aaa ata gtt ggg aac tgg ga 3 ′ ( seq id no : 8 ) and 5 ′ cgt atg agt gag tag aat aaa gtc tta aac tga aca aaa tag a 3 ′ ( seq id no : 9 ) using the plasmid pgps1 . 1 as template dna ( new england biolabs ) and the vector fragment was amplified with primers 5 ′ aag tag ctt ttc tgt gac tgg t 3 ′ ( seq id no : 10 ) and 5 ′ gat ggc atg aca gta aga gct 3 ′ ( seq id no : 11 ) using the plasmid pgps1 . 1 ( new england biolabs ) as template dna . sequencing was performed using the primer 5 ′- gct agt tat tgc tca gcg g - 3 ′ ( seq id no : 5 ). sequencing of tn7 - kan ( stop ) transposon insertion sites in puc19 plasmid was carried out using model 4200 dna sequencer ( li - cor ). sequencing was performed using ird700 - labeled primers 5 ′ agc tgg cga aag ggg gat gtg 3 ′ ( seq id no : 12 ) and 5 ′ tta tgc ttc cgg ctc gta tgt tgt gt 3 ′ ( seq id no : 13 ). aalto , m . k ., jäntti , j ., östling , j ., keränen , s . & amp ; ronne , h . 1997 . mso1p : a yeast protein that functions in secretion and interacts physically and genetically with sec1p . proc . natl . acad . sci . usa 94 : 7331 - 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