CN103849606A - Application of directionally mutated galactosyl transferase - Google Patents
Application of directionally mutated galactosyl transferase Download PDFInfo
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- CN103849606A CN103849606A CN201410027426.6A CN201410027426A CN103849606A CN 103849606 A CN103849606 A CN 103849606A CN 201410027426 A CN201410027426 A CN 201410027426A CN 103849606 A CN103849606 A CN 103849606A
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- galactosyltransferase
- lactose
- orthomutation
- galactosyl transferase
- aminoacid sequence
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Abstract
The invention discloses an application of directionally mutated galactosyl transferase. The research shows that the directionally mutated galactosyl transferase 1 can be changed to only transfer acetyl galactosamine from transferring wild type galactose, and unlike the prior art that the mutated beta1,4-galactosyl transferase 1 can simultaneously transfer galactose and acetyl galactosamine, and the transferring efficiencies of the directionally mutated galactosyl transferase and the beta1,4-galactosyl transferase 1 are both approximate to 50%, the directionally mutated galactosyl transferase 1 and the beta1,4-galactosyl transferase 1 are greatly different from each other. Therefore, the directionally mutated galactosyl transferase 1 can be applied to production of natural milk without lactose or with very low lactose content. The mutated galactosyl transferase 1 can be changed to transfer acetyl galactosamine from transferring galactose, so that the lactose components in milk are changed and the milk is converted into a lactose analog; therefore, novel milk barely containing lactose can be produced by mutating galactosyl transferase 1 genes of mammals by using a mutation breeding technique and lactose intolerance can be avoided.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of application of galactosyltransferase 1 of orthomutation.
Background technology
Along with developing rapidly of genetic engineering technique, we can cultivate various biological new lines by genetic modification means, especially very ripe for the technique means of some model animalss.Now conventional genetic modification means have: transgenic technology, conventional gene Knockout, gene knocks in technology and condition knocks out technology etc.Such as gene is knocked in technology, can in target gene, introduce specifically the gene mutation type of catastrophe point with simulating human familial inheritance disease, also exogenous origin gene integrator can be arrived to genomic specific site, or carry out homogenic substituting between species.But to concrete mutational site and prominent nyctitropic selection, be related to the concrete function of saltant type biology, also need to carry out large quantity research.
Glycosyltransferase catalysis sugar is from the transfer process of donor, and the formation of responsible glycosidic link.Glycosyltransferase has specificity to nucleotide sugar donor and acceptor.β Isosorbide-5-Nitrae-galactosyltransferase 1 is one of the most extensive glycosyltransferase of current people's research.This enzyme expression amount in mammiferous mammary gland is very high, and expresses and increase with lactation process.In cell, galactosyltransferase 1 is mainly distributed on golgi body, and its basic function is to be responsible for galactosyl to be transferred to acetylglucosamine or glucose from saccharide donor UDP-galactoside, forms β-Isosorbide-5-Nitrae glycosidic link.But existing document shows, the β Isosorbide-5-Nitrae-galactosyltransferase 1 after sudden change will shift semi-lactosi and acetylamino galactosamine simultaneously, and both transfer efficiencies are close, and be all 50%
[1].
Summary of the invention
The object of the invention is to knock in technology by gene, a kind of galactosyltransferase 1 of orthomutation is provided.
Another object of the present invention is to provide the application of the galactosyltransferase 1 of this orthomutation.
Object of the present invention can be achieved through the following technical solutions:
The galactosyltransferase 1 of orthomutation is in the application of producing in the seeding milk that does not contain lactose or utmost point lower lactose level, wherein, the galactosyltransferase 1 of described orthomutation is by genetic engineering means orthomutation ox wild-type beta 1, the 289th tyrosine of 4-galactosyltransferase 1 aminoacid sequence, or pig wild-type beta 1, the 288th tyrosine of 4-galactosyltransferase 1 aminoacid sequence, or the tyrosine in other similar sites of Mammals, the transfer galactosyl that makes wild-type to the transfer acetylamino galactosamine that becomes saltant type on acetylglucosamine or glucose on acetylglucosamine or glucose.
Described tyrosine preferred orientation sports leucine.
Ox β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.1.
Pig β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.2.
A kind of production does not contain the method for the seeding milk of lactose or utmost point lower lactose level, to Mammals β 1, the tyrosine of 4-galactosyltransferase 1 aminoacid sequence specific site suddenlys change, the transfer galactosyl that makes wild-type to the transfer acetylamino galactosamine that becomes saltant type on acetylglucosamine or glucose on acetylglucosamine or glucose, thereby make lactose (the Gal β 1 in milk, 4Glc) change and become a kind of material that trace own exists in bovine coloctrum of lactose analogue (GalNAc β Isosorbide-5-Nitrae Glc)
[2].
Utmost point lower lactose level of the present invention refers to and does not substantially contain lactose.
Described Mammals is selected from pig, ox or sheep.
Described method is preferably by the 289th tyrosine of ox wild-type beta Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence, or the 288th tyrosine orthomutation of pig wild-type beta Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence is leucine.
Ox β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.1.
Ox β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.2.
Beneficial effect:
The present invention finds that through research the galactosyltransferase 1 of orthomutation can become and substantially only shift acetylamino galactosamine from the transfer semi-lactosi of wild-type, not as the β 1 after the sudden change of reporting in prior art, 4-galactosyltransferase 1 is all 50% by shift that semi-lactosi and acetylamino galactosamine and both transfer efficiencies be close simultaneously, and our experimental result and existing report make a big difference.
Because the source of lactose is to shift a galactosyl to glucosyl group by β Isosorbide-5-Nitrae-galactosyltransferase 1 in milk, thereby generate lactose.As we β 1 that suddenlyd change, after 4-galactosyltransferase 1, β 1,4-galactosyltransferase 1 just shifts an acetylamino galactosamine to glucosyl group, generate a kind of analogue (GalNAc β 1 of lactose, 4Glc), and no longer shift semi-lactosi, so regeneration not substantially of the lactose in milk.
Based on above-mentioned great discovery of the present invention, and lactose in Mammals milk source relies on to a great extent is galactosyltransferase 1, the present invention proposes the galactosyltransferase 1 of orthomutation in the application of producing in lactose-free seeding milk.Galactosyltransferase 1 after sudden change becomes transfer acetylamino galactosamine from shifting semi-lactosi, so changed the lactose constituent in milk, become a kind of lactose analogue, so can produce and contain novel free oligosaccharides and lactinated Novel milk hardly by mutation breeding technology mammiferous galactosyltransferase 1 gene that suddenlys change, so just can avoid the generation of lactose intolerance.
Brief description of the drawings
Fig. 1 is pig of the present invention source β 4GalT1 and β 4GalT1
mutwestern-blot result figure
Fig. 2 is ox of the present invention source β ' 4GalT1 and β ' 4GalT1
mutwestern-blot result figure
Fig. 3 is pig of the present invention, ox source β 4GalT1 (β ' 4GalT1) and β 4GalT1
mut(β ' 4GalT1Mut) independently adds respectively the LC spectrogram of the reaction system of UDP-Gal and two kinds of substrates of UDP-GalNAc.
Embodiment
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following instance.
Embodiment 1
According to the gene order design primer of pig source β Isosorbide-5-Nitrae-galactosyltransferase 1 in Gene Bank, primer two ends are with base sequence and the protectiveness base of restriction enzyme EcoR1 and Xho1.
P1-F:5'-CGGAATTCCGAGGGGCCGCACCGCAG-3'（SEQ?ID?NO.3）
P1-R:5'-CCGCTCGAGCTAGCTCGGTGTCCCGATGTCCACT-3'（SEQ?ID?NO.4）
Extracting RNA from pork liver, adopts the Trizol test kit of Takara company to carry out the extracting experiment of RNA.The RNA obtaining carries out reverse transcription and obtains cDNA, and reverse transcription test kit is from Promega company.
The PCR test kit that adopts Takara company to provide, taking cDNA as template, P1-F and P1-R carry out pcr amplification.PCR loop parameter: 98 DEG C of denaturations, 5min; 98 DEG C, 40s; 62 DEG C, 1min; 72 DEG C, 1min carries out 35 circulations altogether; 72 DEG C are extended 10min again.
Adopt the glue recovery test kit of Axygen company that goal gene fragment is carried out to purifying.Goal gene after purifying and empty carrier pet30a are carried out respectively to EcoR1 and Xho1 double digestion, respectively the goal gene after double digestion and carrier pet30a are cut to glue recovery, then goal gene and carrier are carried out to conventional ligation.
To connect product and transform intestinal bacteria TOP10.Cell after transforming is coated to the LB agar plate that contains Kana, overnight incubation.Picking colony PCR checking from flat board, the order-checking of positive colony sample presentation, obtains right-on clone strain.The genbank accession number of its gene is GI:545853190.
The plasmid β 4GalT1-pet30a of the bacterial strain that extracting contains correct object fragment, extraction agent box is provided by Axygen company.Plasmid is transformed into and expresses engineering bacteria BL21(DE3).
Embodiment 2
According to the requirement of the Quick-change point mutation test kit of stratagene company, design following primer:
P2-F:5'-CCTTATGTGCAGCTGTTCGGCGGTGTCTCTG-3'（SEQ?ID?NO.5）
P2-R:5'-GACACCGCCGAACAGCTGCACATAAGGGAG-3'（SEQ?ID?NO.6）
The β 4GalT1-pet30a plasmid that the process of PCR builds taking embodiment 1 is template; change the 865-867 bit base TAT of β 4GalT1 gene into CTG; by pig source β 1; the 288th tyrosine of 4-galactosyltransferase 1 (Tyr) sports leucine (Leu); PCR loop parameter: 98 DEG C of denaturations, 1min; 98 DEG C, 50s; 60 DEG C, 50s; 68 DEG C, 8min carries out 18 circulations altogether; 68 DEG C are extended 10min again.The PCR product restriction enzyme Dpn1 obtaining digests original plasmid template, will transform high efficiency competent cell through the PCR product of restriction enzyme Dpn1 digestion.After cultivation, choose bacterium extraction plasmid and check order, can obtain the plasmid β 4GalT1 that contains mutator gene
mut-pet30a.Extracting plasmid β 4GalT1
mut-pet30a translation table expression engineered bacteria BL21 (DE3).
The abduction delivering of embodiment 3 β Isosorbide-5-Nitrae-galactosyltransferases 1 and mutant thereof
By β 4GalT1-pet30a and β 4GalT1 after qualification
mut-pet30a recombinant plasmid transformed is in expressive host E.coliBL21 (DE3).Being placed in 37 DEG C of incubators is cultured to and grows up to sizeable bacterium colony.The transformant that picking contains recombinant plasmid is seeded to respectively the LB liquid medium containing kana (50 μ g/mL), puts 37 DEG C of shaking tables, and 250r/min shakes overnight incubation.Being forwarded to fresh LB(400mL by 1% inoculum size (v/v)) in nutrient solution, 37 DEG C, 250r/min shaking culture is to logarithmic phase (OD
600≈ 0.6-0.8), add respectively inductor IPTG to final concentration 1.0mmol/L, put 25 DEG C of shaking tables, 250r/min abduction delivering 3h.
The purifying of embodiment 4 target proteins
Get the recombinant bacterium nutrient solution that appropriate embodiment 3 methods are carried out abduction delivering, the centrifugal 20min of 4800g/min collects thalline, and thalline is resuspended in lysate 10mL(p H7.550mM NaCl; 50mM Tris-HCl; 1%Triton) in damping fluid, ultrasonication cell in ice bath.By the sample 13000r/min after ultrasonication, 4 DEG C of centrifugal 20min get supernatant and are crude enzyme liquid.
Due to β 4GalT1-pet30a and the β 4GalT1 of design
mut-pet30a expression plasmid expression product N holds with 6 continuous Histidines, can pass through affinity column (Ni-NTA sepharose) affinity purification.First use elutriant balance pillar (pH8.050mM NaCl50mM Tris-HCl), by crude enzyme liquid load upper prop; Adopt 5 times of volume elutriants (pH8.050mM NaCl50mM Tris-HCl10mM imidazoles) to carry out wash-out except foreigh protein removing, then use a certain amount of elutriant (pH8.050mM NaCl50mM Tris-HCl250mM imidazoles) to collect target protein.Preserve target protein, for subsequent experimental.
Embodiment 5 purifying target protein later, does the expression of western-blot checking target protein
Get respectively β 1, purifying target protein 30 μ L at the beginning of two kinds of 4-galactosyltransferase 1 and mutant thereof, add the sample-loading buffer of 5X, make its inactivation at 100 DEG C of heating 10min, adopt the SDS-PAGE of 12% concentration of polyacrylamide to run protein sample, about concentrated glue 80v voltage half an hour, separation gel 100v voltage is about 2 hours, then carry out the transferring film of 2 hours, it is forwarded on nitrocellulose filter, voltage uses 150v.After completing, transferring film carries out the sealing of 1-2 hour.Then the anti-histidine-tagged monoclonal antibody (from Sigma company) that uses mouse source to specifications require overnight incubation, the antibody of the anti-mouse that two anti-employing goats are originated is at room temperature hatched 1-2 hour, finally uses DAB to carry out color reaction.Western-bolt figure is as shown in 1.
Embodiment 6
According to the gene order design primer of ox source β Isosorbide-5-Nitrae-galactosyltransferase 1 in Gene Bank, primer two ends are with base sequence and the protectiveness base of restriction enzyme Kpn1 and EcoR1.
P3-F:5'-GGGGTACCAAGATGAAGTTTCGGGAGCCG-3'（SEQ?ID?NO.7）
P3-R:5'-GGAATTCCTAGCTCGGCGTCCCGATGT-3'（SEQ?ID?NO.8）
Extracting RNA from beef liver, adopts the Trizol test kit of Takara company to carry out the extracting experiment of RNA.The RNA obtaining carries out reverse transcription and obtains cDNA, and reverse transcription test kit is from Promega company.
The PCR test kit that adopts Takara company to provide, taking cDNA as template, P3-F and P3-R carry out pcr amplification.PCR loop parameter: 98 DEG C of denaturations, 5min; 98 DEG C, 40s; 62 DEG C, 1min; 72 DEG C, 1min carries out 35 circulations altogether; 72 DEG C are extended 10min again.
Adopt the glue recovery test kit of Axygen company that goal gene fragment is carried out to purifying.Goal gene after purifying and empty carrier pet30a are carried out respectively to double digestion, and restriction enzyme Kpn1 and EcoR1 are provided by Thermo company.Respectively the goal gene after double digestion and carrier pet30a are cut to glue recovery, then goal gene and carrier are carried out to conventional ligation.
To connect product and transform intestinal bacteria TOP10.Cell after transforming is coated to the LB agar plate that contains Kana, overnight incubation.Picking colony PCR checking from flat board, the order-checking of positive colony sample presentation, obtains right-on clone strain.The accession number of its genbank is GI:31343557.
Plasmid β ' the 4GalT1-pet30a of the bacterial strain that extracting contains correct object fragment, extraction agent box is provided by Axygen company.Plasmid is transformed into and expresses engineering bacteria BL21(DE3).
Embodiment 7
According to the requirement of the Quick-change point mutation test kit of stratagene company, design following primer:
P4-F:5'-CCTTACGTGCAGCTGTTTGGAGGTGTCTCTGCTC-3'（SEQ?ID?NO.9）
P4-R:5'-AGACACCTCCAAACAGCTGCACGTAAGGTAGGCTA-3'（SEQ?ID?NO.10）
β ' the 4GalT1-pet30a plasmid that the process of PCR builds taking embodiment 6 is template; change the 868-870 bit base TAT of β ' 4GalT1 gene into CTG; by ox source β 1; the 289th tyrosine of 4-galactosyltransferase 1 (Tyr) sports leucine (Leu); PCR loop parameter: 98 DEG C of denaturations, 1min; 98 DEG C, 50s; 60 DEG C, 50s; 68 DEG C, 8min carries out 18 circulations altogether; 68 DEG C are extended 10min again.Digest original plasmid template with restriction enzyme Dpn1, will transform high efficiency competent cell through the PCR product of restriction enzyme Dpn1 digestion.After cultivation, choose bacterium extraction plasmid and check order, can obtain the plasmid β ' 4GalT1 that contains mutator gene
mut-pet30a.Extracting plasmid β ' 4GalT1
mut-pet30a translation table expression engineered bacteria BL21 (DE3).
The abduction delivering of embodiment 8 β Isosorbide-5-Nitrae-galactosyltransferases 1 and mutant thereof
By β ' 4GalT1-pet30a and β ' 4GalT1 after qualification
mut-pet30a recombinant plasmid transformed is in expressive host E.coli BL21 (DE3).Being placed in 37 DEG C of incubators is cultured to and grows up to sizeable bacterium colony.The transformant that picking contains recombinant plasmid is seeded to respectively the LB liquid medium containing kana (50 μ g/mL), puts 37 DEG C of shaking tables, and 250r/min shakes overnight incubation.Being forwarded to fresh LB(400mL by 1% inoculum size (v/v)) in nutrient solution, 37 DEG C, 250r/min shaking culture is to logarithmic phase (OD
600≈ 0.6-0.8), add respectively inductor IPTG to final concentration 1.0mmol/L, put 25 DEG C of shaking tables, 250r/min abduction delivering 3h.
The purifying of embodiment 9 target proteins
Get the recombinant bacterium nutrient solution that appropriate embodiment 8 methods are carried out abduction delivering, the centrifugal 20min of 4800g/min collects thalline, and thalline is resuspended in lysate 10mL(p H7.550mM NaCl; 50mM Tris-HCl; 1%Triton) in damping fluid, ultrasonication cell in ice bath.By the sample 13000r/min after ultrasonication, 4 DEG C of centrifugal 20min get supernatant and are crude enzyme liquid.
Due to β ' 4GalT1-pet30a and the β ' 4GalT1 of design
mut-pet30a expression plasmid expression product N holds with 6 continuous Histidines, can pass through affinity column (Ni-NTA sepharose) affinity purification.First use elutriant balance pillar (pH8.050mM NaCl50mM Tris-HCl), by crude enzyme liquid load upper prop; Adopt 5 times of volume elutriants (pH8.050mM NaCl50mM Tris-HCl10mM imidazoles) to carry out wash-out except foreigh protein removing, then use a certain amount of elutriant (pH8.050mM NaCl50mM Tris-HCl250mM imidazoles) to collect target protein.Preserve target protein, for subsequent experimental.
Embodiment 10 purifying target protein later, does the expression of western-blot checking target protein
Get respectively β 1, purifying target protein 30 μ L at the beginning of two kinds of 4-galactosyltransferase 1 and mutant thereof, add the sample-loading buffer of 5X, make its inactivation at 100 DEG C of heating 10min, adopt the SDS-PAGE of 12% concentration of polyacrylamide to run protein sample, about concentrated glue 80v voltage half an hour, separation gel 100v voltage is about 2 hours, then carry out the transferring film of 2 hours, it is forwarded on nitrocellulose filter, voltage uses 150v.After completing, transferring film carries out the sealing of 1-2 hour.Then the anti-histidine-tagged monoclonal antibody (from Sigma company) that uses mouse source to specifications require overnight incubation, the antibody of the anti-mouse that two anti-employing goats are originated is at room temperature hatched 1-2 hour, finally uses DAB to carry out color reaction.Western-bolt figure is as shown in 2.
Embodiment 11
β Isosorbide-5-Nitrae-galactosyltransferase 1(embodiment 4 and 9 preparation of pig source and Niu Yuan) and the zymologic property of mutant measure and carry out altogether 8 group reactions:
37 DEG C of reactions are run LC-MS analytical results in 1 hour later, and result as shown in Figure 3.
Result shows: the pig source of wild-type and ox source β Isosorbide-5-Nitrae-galactosyltransferase 1 can produce lactose, but can not produce lactose analogue GalNAc β Isosorbide-5-Nitrae Glc; β Isosorbide-5-Nitrae-the galactosyltransferase 1 of saltant type can not produce lactose, can only produce lactose analogue GalNAc β Isosorbide-5-Nitrae Glc.
In enzyme biopsy survey, the structural formula of substrate and product is as follows:
Reference:
[1]Ramakrishnan,B.Qasba,P.K.Structure-based?design?of?beta
1,4-galactosyltransferase?I(beta4Gal-T1)with?equally?efficient
N-acetylgalactosaminyltransferase?activity:point?mutation?broadens?beta4Gal-T1donor?specificity.J?Biol?Chem.(2002)20833-9.
[2]Van?den?Nieuwenhof,Ingrid?M,Schiphorst,Wietske?ECM,Van?den?Eijnden,Dirk?H.The?lactose?analog?GalNAcβ1→4Glc?is?present?in?bovine?colostrum:Enzymatic?basis?for?its?occurrence.FEBS?letters.(1999)377-380.
Claims (9)
1. the galactosyltransferase 1 of orthomutation is in the application of producing in the seeding milk that does not contain lactose or utmost point lower lactose level, wherein, the galactosyltransferase 1 of described orthomutation is by genetic engineering means orthomutation ox wild-type beta 1, the 289th tyrosine of 4-galactosyltransferase 1 aminoacid sequence, or pig wild-type beta 1, the 288th tyrosine of 4-galactosyltransferase 1 aminoacid sequence, or the tyrosine in other similar sites of Mammals, make wild-type beta 1, the transfer galactosyl of 4-galactosyltransferase 1 becomes saltant type β 1 on acetylglucosamine or glucose, the transfer acetylamino galactosamine of 4-galactosyltransferase 1 is on acetylglucosamine or glucose.
2. application according to claim 1, is characterized in that described tyrosine orthomutation is leucine.
3. application according to claim 2, is characterized in that ox β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.1.
4. application according to claim 2, is characterized in that pig β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.2.
5. a production is containing the method for the seeding milk of lactose or utmost point lower lactose level, it is characterized in that Mammals β 1, the tyrosine of 4-galactosyltransferase 1 aminoacid sequence specific site suddenlys change, make wild-type beta 1, the transfer galactosyl of 4-galactosyltransferase 1 becomes saltant type β 1 on acetylglucosamine or glucose, the transfer acetylamino galactosamine of 4-galactosyltransferase 1 is on acetylglucosamine or glucose, thereby make lactose (the Gal β 1 in milk, 4Glc) change and become lactose analogue (GalNAc β 1, 4Glc).
6. method according to claim 5, is characterized in that described Mammals is selected from pig, ox or sheep.
7. method according to claim 5, it is characterized in that ox wild-type beta 1, the 289th tyrosine of 4-galactosyltransferase 1 aminoacid sequence, or the 288th tyrosine orthomutation of pig wild-type beta Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence is leucine.
8. method according to claim 5, is characterized in that ox β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.1.
9. method according to claim 5, is characterized in that ox β Isosorbide-5-Nitrae-galactosyltransferase 1 aminoacid sequence after orthomutation is as shown in SEQ ID NO.2.
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| WO2004063344A2 (en) * | 2003-01-10 | 2004-07-29 | Government Of The United States Of America As Represented By The Sercretary Of The Department Of Health And Human Services National Institutes Of Health | CATALYTIC DOMAINS OF β/1,4)-GALACTOSYLTRANSFERASE I HAVING ALTERED DONOR AND ACCEPTOR SPECIFICITIES, DOMAINS THAT PROMOTE IN VITRO PROTEIN FOLDING, AND METHODS FOR THEIR USE |
| WO2005056783A1 (en) * | 2003-12-05 | 2005-06-23 | Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Catalytic domains of beta(1,4)-galactosyltransferase i having altered metal ion specificity |
| WO2009025645A1 (en) * | 2007-08-22 | 2009-02-26 | Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Beta 1,4-galactosyltransferases with altered donor and acceptor specificities, compositions and methods of use |
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| WO2004063344A2 (en) * | 2003-01-10 | 2004-07-29 | Government Of The United States Of America As Represented By The Sercretary Of The Department Of Health And Human Services National Institutes Of Health | CATALYTIC DOMAINS OF β/1,4)-GALACTOSYLTRANSFERASE I HAVING ALTERED DONOR AND ACCEPTOR SPECIFICITIES, DOMAINS THAT PROMOTE IN VITRO PROTEIN FOLDING, AND METHODS FOR THEIR USE |
| WO2005056783A1 (en) * | 2003-12-05 | 2005-06-23 | Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Catalytic domains of beta(1,4)-galactosyltransferase i having altered metal ion specificity |
| WO2009025645A1 (en) * | 2007-08-22 | 2009-02-26 | Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Beta 1,4-galactosyltransferases with altered donor and acceptor specificities, compositions and methods of use |
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| BOOPATHY RAMAKRISHNAN AND PRADMAN K. QASBA: "Structure-based Design of _1,4-Galactosyltransferase I (β4Gal-T1) with Equally Efficient N-Acetylgalactosaminyltransferase Activity", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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