Patent Application: US-200913054815-A

Abstract:
the present invention generally relates to a modified strain of yeast . more specifically , it relates to a strain of pichia pastoris , deficient in its native yeast aquaporin aqy1 or expressing non - functional aqy1 , as well as related methods and uses . in a first aspect , the present invention comprises a strain of the organism p . pastoris that is deficient in aqy1 .

Description:
as used herein , the terms “ aqy1 deficient strain ” or “ deficient in aqy1 ” etc . signify any modified strain of pichia pastoris that does not express the native aquaporin aqy1 or express a non - functional aqy1 . as a person skilled in the art will appreciate , there are several means available to achieve this inventive concept , and the invention should not be construed as limited to the specific examples or enabling disclosure herein . as used herein , the terms “ cell based functional assay ” or “ cell based assay ” should be construed in the broadest sense , and could for example comprise , without limitation , aquaporin assays to screen for inhibitors or verify functionality of new substances without unwanted background disturbance from aqy1 . the term comprises live cell assays , including spheroplast assays , patch clamping , and related whole cell assays that are known in the art . the novel p . pastoris strain has many uses , as is also detailed in the claims . the aqy1 - deficient strain is ideal for studying the structural biology of membrane proteins , since the invention solves the problem of aqy1 co - purifying , accidentally crystallizing “ false leads ”, which would reduce the chances of recovering crystals of the target protein . the structural biology of membrane proteins is of great interest in drug design , and a further application in this regard would be co - crystallisation with potential inhibitor compounds or chemical fragments . the novel strain can also be used as a source for assays . this includes cell based assays as defined above , or using a protein produced in the inventive strain . the purified protein product , uncontaminated by native aqy1 , could be used for liposome , light scattering or nmr - binding assays , as well as for isothermal calorimetry ( itc ) or other biophysical methods . the novel strain can also be used for protein production in general , with the advantage of avoiding the aqy1 contaminant and possibly also achieving better expression levels of the target protein as a result . the strain is particularly suited to express membrane proteins in this manner , especially aquaporins , ion channels , gpcrs etc . the following examples are provided for illustrative purposes only , and shall not be construed as limiting the scope of the present invention . to disrupt aqy1 in pichia pastoris , the gs115 ( his4 ) strain background ( invitrogen ) was used . we created a ppiczb - vector ( invitrogen ) containing aqy1 by extracting the genome from pichia pastoris and amplifying the aqy1 gene with pcr using primers a and b . the anti - sense primer fused the gene with a his6 - tag . furthermore , ecori - and xbai - restriction sites were added which were used to ligate the gene into the ppiczb - vector , thus yielding the ppiczb - his - construct . the aqy1 gene in this construct was disrupted by inserting saccharomyces cerevisiae his4 with its own promoter . the his4 promoter and the open reading frame ( orf ) was amplified by pcr , using primers c and d designed to add psti restriction sites at the ends of the produced dna . ppiczb - aqy1 - his was linearized by digestion using psti cutting in the orf of aqy1 . the linearized vector and the psti digested pcr product of his4 were ligated and transformed into e . coli and transformants were selected for on zeocin containing plates . successful insertion of his4 into the orf of aqy1 in the ppiczb vector was confirmed by pcr ( primers e and f ). this vector was then used as template in a pcr reaction to amplify the aqy1 - his4 - aqy1 construct ( primers f and g ). the pcr product was precipitated and subsequently transformed into p . pastoris gs115 using standard protocols . transformants were selected for histidine prototrophy and verification of the disruption of aqy1 was confirmed by pcr and phenotypic analysis ( cf . below ). cells were grown in 10 ml bmgy at 30 ° c . for 24 hours , 10 od600 units were then transferred to 10 ml bmmy for final od600 = 1 and incubated for 24 hours at 30 ° c . for the freezing and thawing assay , cells were transferred to 1 ml ypd in eppendorf tubes , final od 600 = 0 . 0005 . an aliquot of 50 ul was plated on ypd plates before stress to serve as reference ( 100 % survival ). in total 8 cycles of freezing in liquid nitrogen for 30 seconds and thawing for 3 minutes in water bath at 30 ° c . followed by plating of aliquots on ypd plates were carried out . survival was scored by counting colony forming units ( cfus ). it has previously been shown that wildtype strains can withstand the stress of freezing and thawing better than aqua - porin deletion strains ( tanghe et al ., 2002 ). the reduced number of cfus in the aqy1 deletion strain proves that aqy1 was successfully disrupted . spheroplast assay to confirm reduction of water transport in aqy1 deletion strain pichia pastoris cells , both wildtype x33 cells and the aqy1 deletion strain , were grown for 24 h in 10 ml bmgy in a rotary shaker at 220 rpm and 30 ° c . subsequently , the protein production was induced by growing the cells in 10 ml bmmy for 24 h as above , starting at od 600 = 1 by spinning down the respective amount at 3000 xg , 10 min , 20 ° c . the cells were sedimented at 3000 xg for five minutes at 20 ° c . and resuspended in 1 . 4 ml / g wet cell weight of te - buffer ( 100 mm tris , ph 8 . 0 , 100 mm edta ) and diluted to a final volume of 3 . 5 ml / g wet cell weight with dh 2 o . 2 - mercapto - ethanol (( 1 / 200th of volume )/ wet cell weight ) was added and incubation took place in a rotary shaker at 220 rpm , 30 ° c . for 45 minutes , whereupon the cells were washed two times by sedimentation as above and resuspension in 10 ml s - buffer ( 1 . 2 m sorbitol , 10 mm mes ph 6 . 0 ). subsequently , the cells were resuspended in 10 ml s - buffer supplemented with zymolyase 20t ( seikagaku corp , 50 u / g wet cell weight ) and incubated in a rotary shaker at 150 rpm , 30 ° c . for 30 minutes . the appearance of the spheroplasts was monitored under a microscope , removal of the cell wall was verified by the observation of bursting upon the addition of dh2o ( 1 : 1 ) due to hypo - osmotic shock . the cells were washed three times by sedimenting at 2000 xg for 5 min at 4 ° c . intermittently . finally , the spheroplasts were resuspended in 10 ml s - buffer . change in light scattering at 440 nm was observed upon mixing with equal amounts of 1 . 8 m sorbitol buffer in a stopped - flow spectrophotometer device ( biologic science instruments ). water transport activity measurements using stopped - flow spectrometry from p . pastoris spheroplasts with the aqy1 gene disrupted compared to the wildtype strain ( with endogenous aqy1 ) gave k - values of 0 . 35 and 2 . 6 , respectively . hence the intrinsic water transport rate of p . pastoris has been lowered 7 . 5 times upon the disruption of the aqy1 gene . agre , p ., king , l . s ., yasui , m ., guggino , w . b ., ottersen , o . p ., fujiyoshi , y ., engel , a . and nielsen , s . 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