Patent Application: US-64320006-A

Abstract:
the present invention is directed to proteins of natural origin and materials made therefrom , in particular to threads , fibers , foams and gels derived therefrom . the invention further provides the use of these proteins / threads and materials in the field of technology , biotechnology and / or medicine , in particular in the manufacture of wound closure or coverage systems , suture materials and in the manufacture of replacement materials , preferably artificial cartilage or tendon materials , as well as in other commercial applications .

Description:
the improvement provided herein was based on the idea to express and study the two major protein constituents of araneus diadematus dragline silk simultaneously . since insects belong to the same phylum as spiders , the inventors chose the insect cell line sf9 ( derived from the fall armyworm spodoptera frugiperda ), for the expression of adf - 3 and adf - 4 using baculoviruses as vectors . recombinant baculoviruses were generated containing partial cdnas of adf - 3 and adf - 4 ( 14 ). in order to monitor synthesis , both proteins were provided with a his 6 - tag . to exclude artificial influences caused by the tag , versions without his 6 - tag were also employed . the recombinant viruses were used to infect sf9 cells for production of the spider silk proteins in the cytoplasm . after 3 days of incubation , infected cells were lyzed by sonification and insoluble cell contents were separated from soluble material by sedimentation . the sediment was dissolved in guanidinium thiocyanate ( gdmscn ) prior to analysis by immunoblotting . while a large fraction of adf - 3 was found to be soluble , adf - 4 was almost entirely insoluble three days after infection under the conditions employed ( fig1 a ) and independent from the presence of the his 6 - tag ( fig4 a ). surprisingly , investigating the aggregates in adf - 4 ( seq no : 2 ) expressing cells revealed filaments that coiled throughout the cytoplasm , whereby most of the cells contained only one or few filaments of a uniform width ( fig1 b ). in contrast , cells infected with control viruses or the adf - 3 ( seq no : 1 ) encoding virus never produced such filaments ( fig4 c , d ). immunofluorescence performed on the infected cells using anti - his 6 antibodies showed specific staining of the filaments thus confirming that the filaments were composed of adf - 4 ( fig1 b ). next , the inventors investigated whether adf - 3 and adf - 4 can co - assemble into filaments . the inventors generated a recombinant baculovirus containing both adf - 3 ( seq no : 1 ) and adf - 4 ( seq no : 2 ) under different and independent promoters , using the pfastbacdual donor plasmid . infection of sf9 cells with this virus resulted in synthesis of both proteins and the formation of protein filaments that showed similar appearance in comparison to the filaments formed by synthesis of adf - 4 alone ( fig4 b ). interestingly , filaments assembled in the dual expression system were entirely formed by adf - 4 with no incorporated or stably associated adf - 3 ( fig1 c and data not shown ). in order to study whether the apparent self - assembly is solely based on properties of adf - 4 or whether additional factors or modifications are involved , the inventors created a recombinant baculovirus coding for a secreted form of his 6 - adf - 4 . infection of cells with this virus led to accumulation of adf - 4 in the culture media of the cells ( fig1 d ). immunofluorescence revealed the abundance of adf - 4 containing secretory vesicles at the cell surface of the infected cells ( fig1 e ). strikingly , the inventors did not observe any formation of adf - 4 filaments neither in compartments of the host cells nor in the culture media . silk thread formation generally depends on the protein concentration as well as on additional factors . interestingly the intracellular ph 6 . 3 of sf9 cells corresponds to the ph in the spinning dope prior to silk thread assembly ( 19 ). further factors required for adf - 4 filament assembly in the cytosolic environment remain elusive . investigating the self - assembling properties of adf - 4 in vitro stressed the importance of additional factors . soluble adf - 4 was readily obtained by dissolving filaments in 6 m gdmscn . dissolved adf - 4 rapidly aggregated upon removal of gdmscn by dialysis or dilution . however , the adf - 4 aggregates formed in vitro showed neither fibrillar structures nor did they display the chemical stability of adf - 4 filaments formed inside the sf9 cells ( see below and fig4 e , f ). the above findings indicate the importance of the specific cytosolic environment , which may include additional , so far unresolved , cytoplasmatic factors important for controlled self - assembly . next the inventors characterized the morphology of adf - 4 filaments . the diameters of filaments ranged from 200 nm to 1 μm , however for each single filament the diameter was found to be constant . furthermore , the filaments showed lengths up to 100 μm and often terminated in knots , branches or formed closed circles ( fig2 a , d , e ). filaments displayed a smooth surface and were often associated with nanofibers ( diameter ˜ 5 nm ) and other protein aggregates ( fig2 ). immunoblotting , and immunoelectron microscopy indicated that filaments and associated assembly forms were composed of adf - 4 ( fig2 c , 3 a ). besides adf - 4 no other abundant protein could be detected in filaments as visualized by sds - page analysis followed by silver staining ( fig3 a ). the low number of filaments per cell and the recruitment of almost the entire cellular adf - 4 into the aggregates indicated that self - assembly of adf - 4 in sf9 cells is likely to be a nucleated process , which previously has been also suggested for the silk spinning process of bombyx mori ( 20 ). the size of the filaments formed in the sf9 cells seemed to be constrained by the volume of the cells making them too short for mechanical force measurements typically performed with silk threads ( 21 ). however , the inventors were able to analyze the chemical stability of wet and dry adf - 4 filaments in comparison to natural dragline silk threads of a . diadematus . dragline threads have been reported to be insoluble in many denaturing agents ( 22 ). application of 2 % sodium dodecylsulfate ( sds ) and 8 m urea apparently had no effect on the structure of adf - 4 filaments and dragline threads after 30 s of exposure ( fig3 and data not shown ). immersion of the filaments in 6 m guanidinium chloride ( gdmcl ) did not lead to solubilization of either adf - 4 filaments or dragline threads , although it did lead to swelling of dragline silk . such swelling is likely caused by fibre supercontraction ( 21 ) which has previously been described for spider silks immersed in aqueous solutions and which results from reformation of hydrogen bonds in the amorphous matrix ( 21 ). in contrast to the denaturants mentioned above , a small drop of 6 m gdmscn completely dissolved adf - 4 filaments as well as dragline threads within seconds ( fig3 ). in consequence the inventors conclude that both structures share molecular interactions , which are responsible for chemical resistance to specific denaturants . the cdnas of seq no : 1 and 2 were cloned into pfastbac ™ donor plasmids from invitrogen . sequences coding for peptide tags were provided 5 ′- terminal to the gene fragments . for his 6 - tagged proteins , genes were excised from the host vector using spei / xhoi and ligated with equally digested pfastbac ™ hta . for t7 - taged ( 23 ) proteins , genes were first cloned into pet21 from novagen using xhoi and ecori . the insert including the t7 - tag coding region was then excised with bglii and xhoi and ligated with pfastbac ™ 1 digested with bamhi / xhoi . for co - expressing adf - 3 ( seq no : 1 ) and adf - 4 ( seq no : 2 ), both genes were cloned into pfasbac ™ dual and provided with sequences coding for t7 - and s - tags ( 24 ). the adf - 4 ( seq no : 2 ) gene was excised from pet21 - adf - 4 ( seq no : 2 ) with bglii / xhoi and ligated with pfasbac ™ dual cleaved with nhei / bamhi . two synthetic oligonucleotides ( mwg biotech ) were annealed to provide an s - tag coding sequence , which resulted in double stranded dna with nhei / bamhi - compatible single strand extensions : pet21 - adf - 3 ( seq no : 1 ) was digested with nhei / bamhi to remove the t7 - tag coding region . the vector was then ligated with the s - tag encoding dna . the s - tagged adf - 3 ( seq no : 1 ) was cloned into pfasbac ™ dual - adf - 4 ( seq no : 2 ) using xhoi / xmai . in the dual construct , adf - 3 and adf - 4 were under the control of the independent p10 ( 25 ) and polyhedrin ( 26 ) promoters . the sequence coding for the secretion signal of honeybee melittin was amplified by pcr using the pmibn5 - hisa vector ( invitrogen ) as template and the following primers containing cpoi restriction sites : ( seq id no : 7 ) 5 ′- ccttcc cggtccg ccatgaaattcttagtcaac ( seq id no : 8 ) 5 ′- ccttcc cggaccg ggcatagatgtaagaaat the resulting pcr product was cut with cpoi and ligated into pfastbac ™ hta - adf - 4 ( seq no : 2 ) digested likewise . positive clones were checked for orientation and correctness by sequencing . sf9 ( spodoptera frugiperda ; atcc #: crl - 1711 ) cells were propagated at 27 ° c . in bioinsect - 1 serum - free insect cell culture medium ( biological industries ). sf9 cells were grown either as monolayers on cover slips in 6 well plates or in shaker flasks agitated at 80 rpm . production of recombinant adf - 3 ( seq no : 1 ) and adf - 4 ( seq no : 2 ) containing baculovirus competent e . coli dh10bac cells , containing bacmid ( baculovirus shuttle vector plasmid ) and a helper plasmid , were used to generate recombinant bacmids according to the manufacturer &# 39 ; s protocol ( invitrogen ). insertion of the gene into the bacmid was verified by pcr . sf9 cells were transfected with recombinant bacmid dna using escort transfection reagent ( sigma - aldrich ) in 6 - well plates . the cells were incubated for 5 h at 27 ° c ., rinsed , and incubated for another 72 h . media were harvested , centrifuged , and the virus - containing supernatant was tittered by plaque assays . sf9 cells ( 3 × 10 6 cells / ml ) were infected with the recombinant viruses at various mois ( multiplicity of infection ) ranging from 0 . 1 to 10 . three days post infection ( pi ), cells were harvested by centrifugation at 500 × g for 5 min . cells were resuspended at 1 . 2 × 10 7 cells / ml in 100 mm nacl , 20 mm n -( 2 - hydroxyethyl ) piperazine - n ′-( 2 - ethanesulfonic acid ) ( hepes ), ph 7 . 5 and lyzed by sonification . soluble and insoluble components were separated by centrifugation at 125 , 000 × g for 30 min . for further analysis , pellets were resuspended in 6 m gdmscn and dialyzed against 8 m urea . supernatant and pellet derived from 1 . 5 × 10 5 cells were loaded on 10 % tris - glycine polyacrylamide gels under reducing conditions and blotted onto pvdf membranes ( millipore ). spider silk proteins were detected using a mouse anti - his 6 monoclonal antibody ( sigma - aldrich , 1 : 10 , 000 ) or a mouse anti - t7 monoclonal antibody ( novagen , 1 : 10 , 000 ) and anti - mouse igg peroxidase conjugate ( sigma - aldrich , 1 : 5 , 000 ) as secondary antibody . an s - protein peroxidase conjugate ( novagen , 1 : 5 , 000 ) was used to directly detect s - tagged adf - 3 . cells grown on cover slips at 50 % confluency were infected with adf - 3 ( seq no : 1 ) or adf - 4 ( seq no : 2 ) containing recombinant viruses at moi = 10 . three days pi cells were fixed with methanol at − 20 ° c . cover slips were incubated with mouse anti - his 6 monoclonal antibody ( roche ) at a 1 : 300 dilution followed by texas red conjugated anti - mouse secondary igg at 1 : 500 dilution . cells were observed with an olympus bx51 fluorescence microscope and images were taken with a magnafire sp camera or analyzed by confocal microscopy . cells were resuspended at 1 . 2 × 10 7 cells / ml in 100 mm nacl , 20 mm hepes , ph 7 . 5 and lyzed by adding 2 % w / v sodium dodecylsulfate followed by incubation at 95 ° c . for 5 min . threads were sedimented at 5 , 000 × g followed by washing with 8 m urea and water bidest . purified filaments were resuspended in water bidest and incubated for 3 min on freshly cleaved mica ( afm ) or loaded on thermanox ®) plastic cover slips ( nalgene nunc ) ( sem ). for afm , samples were rinsed with water bidest four times and air - dried prior to contact mode imaging using a multimode spm ( veeco ). for sem , samples were air dried after removal of the solution , vacuum coated with a gold layer and analyzed with a jsm - 5900lv ( jeol ltd .) at 20 kv . for tem ( jeol ltd .) analysis , filaments were adsorbed onto formvar coated grids and negatively stained with uranyl acetate . for immunostaining , fibers were incubated with mouse anti - his 6 antibodies followed by labeling with 18 nm gold - conjugated goat anti mouse igg . to rule out possible influences of the his 6 - tag on filament formation , t7 - tagged adf - 4 was synthesized in sf9 cells . the filament formation of t7 - tagged adf - 4 was apparently indistinguishable to that of his 6 - tagged adf - 4 ( fig4 a ). thread formation in adf - 3 ( seq no : 1 ) and adf - 4 ( seq no : 2 ) co - expressing cells in sf9 cells co - expressing adf - 3 and adf - 4 , filaments could be detected that displayed an apparently indistinguishable morphology in comparison to filaments formed in cells producing only adf - 4 ( fig4 b ). although immunocytochemistry revealed fluorescent foci in adf - 3 ( seq no : 1 ) expressing cells , filament - like structures could not be observed ( fig4 c , d ). importantly , adf - 3 synthesized in sf9 cells was largely soluble . therefore foci formation represented sub - cellular accumulation rather than protein aggregation . adf - 4 aggregated upon removal of denaturants by dialysis or after dilution into aqueous buffers . the resulting aggregates did not display any fibrillar morphology ( fig4 e ). testing chemical stability revealed that in contrast to adf - 4 filaments , formed in the cytosol , the aggregates formed in vitro were soluble in 2 % sds or 8 m urea ( fig4 f ). the following experiments were performed to demonstrate that proteins derived from spider silk sequences adf - 3 ( seq id no : 1 ) or adf - 4 ( seq id no : 2 ) can be assembled into morphological distinct forms . proteins based on adf - 3 and adf - 4 were constructed , produced and prepared in aqueous solutions as described in biochemistry 2004 vol . 43 pp . 13604 - 11362 ( 27 ). if not otherwise mentioned protein solutions contained 10 mm tris -( hydroxymethyl )- aminomethan ( tris ) ph 8 . 0 . protein spheres displaying diameters ranging between 0 . 5 and 2 μm ( fig5 a ) were generated by adding 0 . 8 m ammonium sulphate to a 0 . 2 % ( w / v ) adf - 4 based protein solution . nanofibrils displaying diameters between 0 . 7 and 4 nm ( fig5 b ) were formed by incubating a 1 % ( w / v ) adf - 4 based protein solution at room temperature for 2 weeks . for the formation of microfibrils 5 - 10 μl of a 25 % ( w / v ) adf - 3 based protein solution was slowly injected into 0 . 5 m potassium phosphate ph 8 . 0 , forming a stable drop of protein solution . after incubation for 1 min the protein drop was removed from the solution using tweezers . after an additional incubation time of 1 min in air a protein fibril could be drawn from the protein drop at a rate of approximately 2 cm / s using a second set of tweezers . the fibrils displayed a round cross section with a diameter of 4 μm ( fig5 c , d ). protein foams ( fig5 e , f ) were generated from solutions containing 2 . 5 mm ammonium peroxodisulfate ( aps ), 100 μm tris ( 2 , 2 ′- bipyridyl ) dichlororuthenium ( ii ) ( rubpy ) and 10 % ( w / v ) adf - 3 based protein or 2 % ( w / v ) adf - 4 based protein . the protein solutions were frothed up with air . to stabilize the resulting foam structure proteins were crosslinked by exposition to visible light from a tungsten lamp for 1 min ( 28 ). foams were subsequently dried at 95 ° c . adf - 4 based nanofibrils at 1 % ( w / v ) concentration ( see section 2 ) displayed a gel like appearance which easily could be disrupted by agitation or shearing . to improve the mechanical properties of the gel aps and rubpy were allowed to enter the gel by diffusion to yield final concentrations of 10 mm aps and 100 μm rubpy . after light induced crosslinking ( see section 4 ) dimensionally stable gels could be obtained ( fig5 g ). adf - 3 derived protein was dissolved in 1 , 1 , 1 , 3 , 3 , 3 - hexafluor - 2 - propanol at 20 mg / ml concentration . 200 μl of the solution were spread on a polystyrene surface ( ca . 20 cm 2 ). after evaporation of the solvent a transparent protein film could be pulled off ( see fig6 ). 1 . j . m . gosline , p . a . guerette , c . s . ortlepp , k . n . savage , j . exp . biol . 202 pt 23 , 3295 - 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