Patent Application: US-201314141890-A

Abstract:
the invention is drawn to a method for purifying membrane - bound proteins expressed in recombinant insect cells using n - laurosarcosine . the invention is particularly suited for expressing cadherin - type receptors cloned from ostrinia nubilalis , european corn borer , and expressed in sf9 insect cells . the method is optionally adapted for use with 6 - his tag proteins .

Description:
ostrinia nubilalis , european corn borer ( ecb ), is a well known pest of zea mays , and cry toxins , in a variety of forms , have been used for decades to combat this pest . it is well within the ordinarily skilled artisan &# 39 ; s purview to introduce and express foreign genes in sf9 insect cells . see , for example , references [ 5 ] and [ 6 ]. dna sequences encoding ecb - cadherins have been described [ 7 ] that includes the aminopeptidase 1 . this novel method is exemplified by purifying two different membrane receptor proteins ; a protein of the cadherin family , and an aminopeptidase , both from the midgut of ostrinia nubilalis , that were expressed in sf9 cells . the receptor proteins were purified to greater than 90 % as judged by sds - page , and their activity was retained as confirmed by measuring their enzymatic activity in functional assays and their ability to bind bt toxins . the binding affinity of the core crylac type bt toxin was measured using surface plasmon resonance spectroscopy and the obtained dissociation constant are determined to be in the specific binding range as stated in the literature . using routine methods , sf9 insect cells were transformed to express either ecb cadherin or ecb apn1 , with or without a 6 - his tag . after the cells were transformed and selected for transgenic expression , they were grown , harvested , and then frozen . frozen sf9 cells expressing the membrane receptors were resuspended in 50 mm caps or tris ( ph 10 . 5 or 8 . 0 ), 300 mm nacl , 1 . 0 % ( w / v ) n - lauroylsarcosine , 0 . 1 mm dtt , and 1 . 0 mm pmsf . after 30 minutes incubation on ice , they were lysed with sonication , then ultra - centrifuged for one hour at 30 , 000 rpm (− 105 , 000 g ). the supernatant was dialysed three hours against 25 mm tris , ph 8 . 0 , 50 mm nacl , 0 . 15 % ( w / v ) n - lauroylsarcosine , 1 mm pmsf , at the volume ratio ( supernatant : buffer ) of 1 : 2 and slow stiffing . the buffer was changed at the ratio of 1 : 10 for another three hours dialysis with the same slow stirring . for proteins tagged with 6 - his , 10 mm imidazole was added to the dialysate , and loaded on 5 ml histrap hp ® column ( sigma - aldrich ) precharged with nickel and pre - equilibrated with 25 mm tris , ph 8 . 0 , 0 . 15 m nacl , 0 . 15 % n - lauroylsarcosine . the column was washed with 50 ml washing buffer ( 25 mm tris , ph 8 . 0 , 0 . 15 m nacl , 0 . 15 % n - lauroylsarcosine , 10 mm imidazole ). the bound proteins were eluted with elution buffer ( 25 mm tris , ph 8 . 0 , 0 . 15 m nacl , 0 . 15 % n - lauroylsarcosine , 0 . 5 m imidazole ). for proteins not labeled with 6 - his , the dialysate was loaded on a 5 ml - hitrap q ® column ( sigma - aldrich ) pre - equilibrated with loading buffer ( 20 mm tris , ph 8 . 0 , 50 mm nacl , 0 . 15 % n - lauroylsarcosine , 0 . 5 mm edta , 1 mm pmsf ). the column was washed with 50 ml washing buffer ( 20 mm tris , ph 8 . 0 , 150 mm nacl , 0 . 15 % n - lauroylsarcosine , 0 . 5 mm edta , 1 mm pmsf ). the protein was eluted with linear gradient : nacl from 150 mm to 725 mm in 20 mm tris , ph 8 . 0 , 0 . 15 % n - lauroylsarcosine , 0 . 5 mm edta , 1 mm pmsf . for increased purity , the protein containing fractions were concentrated and further separated with a superose ® 6 10 / 30 column ( amersham biosciences ). the running buffer was 20 mm tris , ph 8 . 0 , 150 mm nacl , 0 . 15 % n - lauroylsarcosine , 0 . 5 mm edta , 1 mm dtt . 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