Patent Application: US-16212702-A

Abstract:
variable domain murine t - cell receptor genes have been isolated and used to construct cloning and expression vectors . v α , v β , and single chain v α - v β fragments have been expressed as secreted domains in escherichia coli using the vectors . the domains are secreted into the culture supernatant in milligram quantities . the single domains and the single chain t - cell receptors are folded into β - pleated sheet structures similar to those of immunoglobulin variable domains . the secreted fragments may be useful for immunization to generate anti - clonotypic antibodies , in vaccination or for high resolution structural studies . the genes encoding these domains may also serve as templates for in vitro mutagenesis and improvement of affinities of the tcr fragments for their interaction with cognate peptide - mhc complexes .

Description:
the present invention concerns the cloning and expression of the variable domains of a murine t - cell receptor ( tcr ) in a gram - negative bacterial host . the genes encoding the tcr for v α and v β domains of t - cell hybridoma 1934 . 4 ( wraith et al ., 1989 ) were isolated using the polymerase chain reaction ( saiki et al ., 1988 ) and ligated into expression vectors constructed from puc119 ( viera et al ., 1987 ). puc119 contains a lacz promoter sequence upstream of the site into which the v α and v β domain genes were ligated . the new expression vectors included a pelb leader segment in translational frame with the cloned variable domain genes . modification of these expression vectors , containing v α and v β . single variable domains , was accomplished by cloning a v β . encoding segment 3 ′ to a v α gene segment and incorporating a single chain linker peptide . bacterial hosts transformed with this construct were capable of expressing single chain heterodimeric tcr v α v β domains . the tcr genes employed were derived from a pathogenic cd4 + t - cell clone associated with induction of experimental allergic ( autoimmune ) encephalomyelitis ( eae ) in the h - 2 u mouse ( wraith et al ., 1989 ). eae is a prototypic model of t - cell mediated autoimmune disease and may be a valuable model for multiple sclerosis in humans . vα and v β genes derived from 1934 . 4 cells were cloned into vhnco - poly - tag1 to generate v α pelbtag1 ( v α gene only ) and v β pelbtag1 ( v β gene only ) and transformed into e . coli host cells . the nucleotide sequences of the constructions were confirmed by dna sequencing prior to growing up and inducing e . coli recombinants for expression . culture supernatants were analyzed by western blotting which clearly showed that the v 60 and vβ domains were expressed individually and could be secreted into the culture supernatant . the molecular weights , from sds gel analysis , were estimated as 17 kda ( v α - tag1 ) and 14 . 5 kda ( v β - tag1 ). for the v α domain this is significantly higher than that predicted by amino acid analysis , but is similar to the anomalously low gel mobilities observed for single antibody vh domains . the level of secretion of the v α domain was particularly high and was similar to , if not greater than , that reported for immunoglobulin fvd1 . 3 fragment expressed and secreted from e . coli ( ward et al ., 1989 ). the level was estimated at 10 mg per liter of culture , by comparison with culture supernatants of e . coli recombinants harboring psw1 - vhd1 . 3 - vkd1 . 3 - tag1 using western blotting . the relatively high expression level of the v α domain may reflect a propensity of this domain to form homodimers . such homodimer formation could mask the hydrophobic residues of the v α domain which , in a native tcr , interact with analogous vβ residues during v α : v β pairing , thus increasing the solubility ( and secretion levels ) of the homodimer . in contrast to the v α domain , the v β domain was secreted into the culture supernatant at levels of about 0 . 5 - 1 mg per liter of culture , although the intracellular / periplasmic levels of the v β domain were similar to those of the v α domain . the v β protein apparently does not fold into a soluble form as readily as the v α domain . it is expected that the amount of secreted v β will be increased by altering the induction conditions . alternatively , higher levels of soluble vβ domain may be obtained by osmotically shocking the recombinant e . coli cells , followed by denaturation and refolding of the released v β protein . as illustrated in example 2 , the v α and v β domains may be co - expressed and secreted from e . coli recombinants harboring v α v β pelbtag1 . the v α polypeptide was secreted in excess over the v β domain , indicating that at least some of the recombinant tcr protein is not heterodimeric . however , v α domain secretion levels were lower when co - expressed with the v β polypeptide than when expressed and secreted as a single domain . this may be due , for example , to limitations on the amount of protein which can be secreted into the e . coli periplasm , i . e ., v β secretion may compete with v α secretion . alternatively , there may be some polarity effects on the expression of the v α domain , which is 3 ′ to the v β gene in v α vβpelbtag1 . the present invention demonstrates that tcr v α and v β polypeptides can be expressed and secreted from recombinant e . coli cells as either individual domains or co - expressed . the secretion system may be employed as a rapid and economically favorable alternative to existing methods for the production of tcrs or tcr - immunoglobulin chimeras in mammalian cell transfectomas . for purification , the v α and v β domains were expressed with carboxy terminal ( his ) 6 tags . as a preferred method of purification , induction conditions were established allowing isolation of the protein from the periplasmic space using osmotic shock . the osmotic shock fractions were dialyzed against phosphate buffered saline overnight at 4 ° c . with 3 changes , and the dialysate passed through an ni 2 + - nta - agarose column . alternatively , longer induction times were used and the protein purified from the culture supernatant . concentration of the supernatant was then effected by concentration under high pressure using an amicon filtration unit followed by overnight dialysis against pbs and passage through a ni 2 + - nta - agarose column . using purification from osmotic shock fractions , yields of 1 - 2 mg / l v α domain and 0 . 1 - 0 . 2 mg / l v β were obtained . the v α and v β domains do not associate when co - expressed within the same bacterial cell . to drive the association of the two domains , therefore , the v α domain was linked to the v β domain by a synthetic linker and the two domains expressed as a heterodimeric sctcr fragment . this heterodimer may be expressed with carboxy - terminal ( his ) 6 peptide tags and purified using affinity purification on ni 2 + - nta - agarose columns . the purification yields from osmotic shock fractions were 0 . 5 - 1 mg / l culture . to assess the folded state of the recombinant tcr fragments , cd spectral analyses were carried out on the fragments and on the d1 . 3 single chain fv fragment . the minima in the curves at 218 nm for these proteins indicate the presence of a high proportion of β - pleated sheet structure ( johnson , 1990 ). these spectra also indicated a lack of α - helical regions , since a - helical regions result in minima at ˜ 208 nm and 224 nm , and this is consistent with the proposed structural models for tcr extracellular domains ( novotny et al ., 1986 ; chothia et al ., 1988 ), and the structure of the crystallographically solved d1 . 3 fv fragment ( bhat et al ., 1990 ). the maxima at approximately 205 nm in the spectra of the v α domain and the d1 . 3fv fragment has previously been associated with the presence of an abundance of β - turns . overall and in general terms the invention shows that single v α , v β domains and single chain heterodimeric tcrs ( sctcrs ) derived from an encephalitogenic t cell hybridoma may be expressed and purified in yields ranging from 0 . 1 - 2 milligrams per liter of bacterial culture . in addition , structural analysis using cd indicates that the recombinant tcr fragments contain a high proportion of β pleated sheet structures . although molecular modelling has indicated that the extracellular domains of tcrs may resemble immunoglobulin fv and fab fragments in structure ( novotny et al ., 1986 ; chothia et al ., 1988 ), to date this has not been demonstrated empirically . the ability of the v α , v β domains and heterodimeric sctcr to inhibit the binding of the 1934 . 4 t cell hybridoma to cognate peptide - mhc complexes ( n - terminal residues 1 - 11 of myelin basic protein associated with the mhc class ii protein i - a u ; wraith et al ., 1989 ) is of particular interest because it would demonstrate functional activity of the recombinant proteins . it is conceivable , however , that soluble tcr fragments are ineffective inhibitors of the multivalent , high avidity , interaction ( harding and unanue , 1990 ) of t cell borne antigen receptors with peptide - mhc complexes . the tripartite interaction of ‘ native ’ tcr on cd4 + t cells with peptide - mhc complexes may be stabilized by contacts between cd4 residues and the mhc class ii molecule ( sleckman et al ., 1987 : fleury et al ., 1991 ). the absence of this ‘ co - receptor ’ in the recombinant tcrs may therefore decrease the avidity of the interaction further . bacterial strains and plasmids e . coli bmh 71 - 18 was used as host for the cloning and expression of tcr domains ( rüther et al ., 1981 ). plasmid pswi - vh - poly - tag1 ( ward et al ., 1989 ) was modified by replacing puc19 with puc119 ( viera et al ., 1987 ) as the backbone vector . in addition , an ncoi restriction site was inserted into the pelb leader sequence using site directed mutagenesis to generate v h nco - poly - tag1 . the v α and v β genes were isolated from 1934 . 4 hybridoma cells ( dr . d . wraith , cambridge university , department of pathology , immunology division , level 3 laboratories block , addenbrookes &# 39 ; s hospital , hills road , cambridge cb2 2qq , uk ) using a pcr amplification method . 10 6 cells were washed once in sterile pbs , then resuspended in 1 ml sterile deionized water and heated at 100 ° c . for 5 mins . this results in isolation of genomic dna . debris was pelleted by centrifugation for 3 minutes at room temperature at 11 , 000 rpm and 2 - 10 μl of supernatant used in a pcr reaction with the following v α or v β specific primers : v α : i : 5 ′- atc ctt cca tgg ccg act cag tga ctc aga cgg aag gt - 3 ′ ii : 5 ′- aag gat ggt gac cgg ttt att ggt gag ttt ggt tcc - 3 ′ v β : iii : 5 ′- atc ctt cca tgg ccg agg ctg cag tca ccc aaa gtc ca - 3 ′ iv : 5 ′- aag gat ggt gac cag aac agt cag tct ggt tcc tga - 3 ′ for each domain , the oligonucleotides encode either an ncoi or bsteii fragment ( underlined ) to allow restriction enzyme digestion of the pcr products , followed by gel purification using “ geneclean ” ( bio 101 , valley park , mo . 63088 ) and ligation as an ncoi - bsteii fragment into vh h nco - poly - tag1 . cycling conditions were 94 ° c . for 0 . 5 min , 55 ° c . for 0 . 5 min , 72 ° c . for 1 min with taq polymerase added at the end of the first cycle , that is , at 72 ° c . thirty cycles of pcr were conducted and an additional 3 units of taq polymerase added after 15 cycles to minimize occurrence of pcr errors . alternatively , less error - prone polymerases such as vent ™ polymerase ( new england biolabs , beverly , mass . 01915 - 5599 ) may be used . the following examples are intended to illustrate the practice of the present invention and are not intended to be limiting . although the - invention is demonstrated with variable murine t - cell v α and v β domains , other domains will be adaptable to similar constructs as those described hereinabove . likewise , a variety of tags , linker sequences and leader sequences may be employed depending on the particular purification or isolation methods desired to obtain the polypeptide products . the following example illustrates the construction of plasmids for expression of the t - cell receptor single domains , v α and v β and the single chain v α v β construct . two types of plasmids are illustrated ; one with a c - myc tag and the other with a ( his ) 6 tag . other tags could be used . the tag portion for the plasmid construct used in this example is c - myc with the following nucleic acid sequence : gaa caa aaa ctc atc tca ga aga gga tct gaat ′ encoding the following 11 - mer : glu gln lys leu ile ser glu glu asp leu asn . the polylinker sequence is : ctg cag tct aga gtc gac ctc gag ggt cacc . pswi - vh - poly - tag1 ( ward , et al ., 1989 ) was modified by the insertion of a unique ncoi site into the pelb leader sequence using site - directed dideoxynucleotide mutagenesis ( carter , et al ., 1985 ) and the oligonucleotide 5 ′- ggc cat ggc tgg ttg gg - 3 ′ to generate vh nco - poly - tag1 . the pelb leader sequence was atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc gct gcc caa cca gcg atg gc . the underlined portion was converted to ccatgg by mutagenesis . the v α gene isolated and tailored by the pcr was then cloned in translational frame as an ncoi - bsteii fragment into vh nco - poly - tag1 to generate v α pelbtag1 as shown in fig3 . dideoxynucleotide sequencing was carried out to confirm the dna sequences of the plasmid construction . the v β pelbtag1 plasmid was constructed according to the procedure for the v α plasmid except that the v β gene was used in place of v α . the construct is shown in fig3 . to construct the v α v β pelbtag1 plasmid , v α pelbtag1 was modified by replacement of the 5 ′ hindiii site of puc119 ( viera and messing , 1987 ) with an ecori site by ligation of oligonucleotide v . 5 ′- agc tga att c 3 ′ as a duplex into hindiii restricted v α pelbtag1 ( with the ecori site shown underlined ). the ligation destroyed the hindiii site . it was then cloned as an ecori fragment into ecori restricted v β pelbtag1 , shown in fig3 . recombinants were analyzed for correct orientation of the v α gene with respect to the v β gene by restriction enzyme analysis . dideoxynucleotide sequencing was carried out to confirm the dna sequences of the plasmid construction . the v α and v β domains do not associate when they are co - expressed within the same bacterial cell . to drive the association of the two domains , therefore , the v α domain was linked to the v β domain by a synthetic peptide linker and the two domains expressed as a heterodimeric sctcr fragment . this heterodimer was expressed with carboxy - terminal ( his ) 6 peptide tags and purified as herein described for single chain domains . purification yields were 0 . 5 - 1 . 0 mg / l culture . the plasmid v α v β pelbmyc2 ( fig1 ) was constructed in a similar manner to v α pelbmyc or v α v β pelbtag1 , except that the v β pelbmyc gene was cloned 3 ′ to the v α pelbmyc gene . the plasmid scv α v β pelbhis , shown schematically in fig8 was constructed as indicated in fig1 . the v α gene was ligated 5 ′ to the v β gene so that in the expressed protein the v α domain was located at the n - terminus of the v α v β heterodimer . since the v α domain is more soluble than the v β domain and expressed at higher levels , this orientation of the two domains with respect to each other appears to assist in the secretion and folding of the sctcr . the single chain linker , ( gly 4 ser ) 3 ( huston et al ., 1988 ) was ligated into bsteiii - psti restricted v α δv β pelb as in the following dna duplex : to construct sc v α v β pelbmyc , the resulting hindiii - psti fragment ( scv α δv β pelb ) encoding the pelb leader , the v α gene , the single chain linker and the 5 ′ end of the v β gene was ligated into hindiii - psti restricted v β pelbmyc to replace the pelb leader . to insert the ( his ) 6 peptide tag into scv α v β pelbmyc , the plasmid was restricted with bsteii and the following duplex ligated into the construct : 5 ′- gtc acc cat cac cat cac cat cac taa taa - 3 ′ 3 ′ g gta gtg gta gtg gta gtg att att cag tg - 5 ′ recombinant clones with the correct orientation of the ( his ) 6 tag were identified by pcr screening . ligation of the duplex in the correct orientation into bsteii cut scv α v β pelbmyc removed the 3 ′ bsteii site . in addition , the presence of 2 stop codons at the 3 ′ end of the histidine codons prevented readthrough into the downstream c - myc tag sequences . nucleic acid and derived amino acid sequences of the single chain tcr with attached ( his ) 6 tag is shown in fig7 . single stranded dna was purified from extruded phage using polyethylene glycol precipitation . sequencing reactions were then carried out using aliquots of the single stranded dna , appropriate oligonucleotide primers and sequenase ( usb corp , cleveland , ohio 44122 ) as polymerase . random low level incorporation of dideoxynucleotides corresponding to each nucleotide position in the gene which was being sequenced occurred by using low levels of chain terminators ( dideoxynucleotides ) in the reaction mixes . the extended , prematurely terminated single stranded dna molecules were then analyzed by electrophoresis followed by autoradiography with radiolabeled nucleotides included in the reactions to improve the sensitivity of detection . v α pelbhis and v β pelbhis were constructed using the strategy shown in fig1 . prior to expression analysis , all dna constructs were sequenced using the dideoxynucleotide method . single stranded dna was isolated from the clones by growth of the recombinant cells in the presence of helper phage , vcsm13 ( stratagene , la jolla , calif . 92037 ). nucleic acid and derived amino acid sequences for v α pelbhis and v β pelbhis are shown in fig5 and 6 respectively . v α pelbhis was constructed using the strategy outlined in fig1 . this involved restriction by bsteii to remove the single chain linker sequence and the v β domain gene followed by religation . v β pelbhis was constructed using the strategy outlined in fig1 . a psti - ecori fragment encoding the majority of the v β domain gene and the ( his ) 6 tag was isolated following restriction enzyme digestion . this was then ligated into psti - ecori restricted v β pelbmyc to replace the majority of the v β gene and the c - myc tag . a vector scv α v β pelbhis ver . 2 has been constructed in which two codons ( val - thr ) which are located between the 3 ′ end of the jα gene and the 5 ′ end of the ( gly4ser ) 3 linker have been removed , fig4 . these codons are derived from the 3 ′ end of an immunoglobulin heavy chain variable domain , and may therefore interfere with the sctcr structure in the protein expressed from v α v β pelbhis . the two codons were removed using pcr mutagenesis , as shown in fig4 and the following primers . primer a : 5 ′ gta tct gca ccc tcc gat ccg cca ccg ccg cat cca cct 3 ′ primer b : 5 ′ atc agc atc cac ctc cgc ctg aac cgc ctc cac ccc gtt taa tgg 3 ′ the sctcr encoded by scv α v β pelbhis ver . 2 can be secreted and purified in yields of 0 . 5 - 1 mg / liter of culture , and cd analysis indicates that the protein is folded into a similar , if not the same , structure as that encoded by sc v α v β pelbhis . thus , for practical purposes of sc tcr production the two constructs do not appear to differ . the nucleic acid and derived amino acid sequence of the single chain construct is shown in fig9 . the following are examples of expression of v α , v β and v α v β t - cell receptor domains employing e . coli hosts transformed with the vectors of example 1 . detection of unpurified recombinant protein in culture supernatants or osmotic shock fractions was performed as follows : [ 0108 ] e . coli recombinants harboring v α pelbmyc , v β pelbmyc v α v β pelbmyc , or scv α v β pelbmyc were grown up in 2 × ty ( or 4 × ty ) plus 100 μl ampicillin and 1 % ( wt : vol ) glucose to early stationary phase , pelleted by centrifugation , washed once in either 2 × ty ( or 4 × ty ) or 50 mm nacl and then induced by resuspension in 2 × ty ( or 4 × ty ) plus 100 μg / ml ampicillin and 1 mm isopropyl - β - d - thiogalactopyranoside ( iptg ) for 14 - 16 hrs . cultures were grown and induced at 37 ° c . with shaking at 250 rpm . culture supernatants were analyzed by western blotting ( towbin et al ., 1979 ; ward et al ., 1985 ) using monoclonal antibody 9e10 ( evan et al ., 1985 ) followed by anti - mouse f c conjugated to horseradish peroxidase ( icn immunobiologicals ) for detection . diamino benzidine ( sigma , st . louis , mo .) was used as the horseradish peroxidase substrate . to detect expressed proteins in osmotic shock fractions the following procedure was followed . recombinant cells harboring v α pelbmyc , v β pelbmyc , v α v β pelbmyc or scv α v β pelbmyc were grown up at 30 ° c . for 12 - 16 hours in the same media as above , pelleted by centrifugation and washed in either 4 × ty or 50 mm nacl , and resuspended in 4 × ty plus 100 μg / ml ampicillin plus 0 . 1 mm iptg plus 1 mg / ml leupeptin ( a protease inhibitor ) and 10 μg / ml pmsf for 5 - 6 hours . periplasmic fractions were isolated using cold tes buffer or 20 % tes as described below . osmotic shock fractions ( see below ) were then analyzed using western blotting and the 9e10 monoclonal antibody as above . fig2 shows the results of western blotting for v α , v β and scv α v β domains tagged with carboxy terminal c - myc . for optimal yields of purified t - cell receptor proteins , recombinants harboring v α pelbhis , v β pelbhis and v α v β pelbhis were employed . 1 - 2 liter cultures of recombinants were grown up in 4 × ty media ( double strength 2 × ty ) plus 100 pg ampicillin / ml plus 1 % ( w / v ) glucose for 15 hr at 30 ° c . cells were pelleted by centrifugation , washed once in 4 × ty and resuspended in 1 liter of 4 × ty plus 100 μg ampicillin / ml , 0 . 1 mm - iptg , 1 μg leupeptin / ml and 10 μg pmsf / ml and grown at 25 ° c . for 5 - 5 . 5 hrs . at this stage the majority of the recombinant protein was located in the periplasm , and was isolated by osmotically shocking the cells as follows : cells were cooled by standing on ice for 10 minutes , and then pelleted by centrifugation ( 6000 rpm , 15 minutes at 4 ° c .). cell pellets were resuspended in cold ( at 0 - 4 ° c .) 200 mm tris - hcl ph 8 . 0 , 500 mm sucrose and 0 . 5 mm na 2 edta ( tes , 40 mls used per 1 liter culture ). cells were incubated in tes for 20 - 40 minutes at 0 ° c . ), and then pelleted by centrifugation ( 10 , 000 rpm , 10 minutes at 4 ° c .). the supernatant was dialyzed against phosphate buffered saline overnight ( 3 changes at 4 ° c .). the pellets were resuspended in cold 20 % v / v tes and incubated for 20 - 40 minutes at 0 ° c . the cells were again pelleted by centrifugation ( 10 , 000 rpm , 10 minutes at 4 ° c . ), and the supernatant was dialyzed against phosphate buffered saline overnight ( 3 changes at 4 °). both tes and 20 % tes supernatants were then passed through ni 2 + - nta - agarose columns . bound protein was batch eluted in 1 - 2 ml fractions with 250 mm imidazole , ph 9 . 2 . to reduce non - specific binding of additional proteins , the column was washed with 500 mm nacl / 100 mm tris hcl , ph 8 , and the same at ph 7 . 4 , prior to elution . the purified protein was dialyzed extensively against 10 mm nah 2 po 4 , ph 7 . 0 , prior to cd analysis . purity of t - cell receptor fragments was assessed by 15 % sds / polyacrylamide gel electrophoresis followed by staining with coomassie brilliant blue . the sds - page stained gel of the purified proteins is shown in fig1 . yields of the purified v α and v β domains were approximately 1 - 2 mg / l culture and 0 . 2 mg / l culture respectively . crosslinking experiments with dithiobis ( succimidylpropionate ) ( dsp ) indicated that the v α domain tended to form homodimers . to analyze the oligomeric state of the v α domain , the purified protein ( at a concentration of approximately 1 mg / ml in pbs ) or culture supernatant from induced cultures ( see above ; 24 hours or more induction ) was used . the purified protein was v α ( his ) 6 ( carboxy terminal ( his ) 6 tag ) and the culture supernatant contained v α myc ( carboxy terminal c - myc tag ). 50 μl of each sample was incubated with 0 . 1 - 2 mm of dsp for one hour at room temperature . the crosslinked samples were then analyzed by sds - page ( under non - reducing conditions ) followed by either staining with coomassie brilliant blue ( for v α ( his ) 6 ) or western blotting ( for v α myc ). for the western blotting , the v α domain was detected using the 9e10 monoclonal antibody as above . for a significant proportion of v α domain , the size following incubation with crosslinker was approximately 30 kda , indicating the formation of homodimers . as a single chain polypeptide , the 1934 . 4 hybridoma cell - derived sctcr with a ( his ) 6 peptide tag was secreted into the periplasm and purified using ni 2 + - nta - agarose in yields of about 0 . 5 - 1 . 0 mg / l culture . the lower growth and induction temperature and lower iptg concentration ( 0 . 1 mm ), about 25 ° c ., was particularly beneficial in inducing higher expression yields for the single chain v α v β heterodimer . an alternative purification of the single chain v α v β domain employed an antibody - linked sepharose column . the purification of the sctcr by affinity chromatography indicated epitopic recognition by the antibody employed , monoclonal antibody kj16 which is specific for murine v βγ ( kappler et al ., 1988 ). the following example illustrates that the expression vectors of example 1 are not limited to expression in e . coli . serratia marcescens was employed as host in the following example . expression and secretion of tcr single chain tcrs from s . marcescens the plasmid scv α v β pelbhis was transformed into s . marcescens by electroporation and transformants selected on 2 × ty agar plates with 1 mg / ml ampicillin and 1 % w / v glucose , or minimal media ( sambrook et al ., 1989 ) plates plus 1 mg / ml ampicillin plus 1 % w / v glucose . transformants were grown up in minimal media plus 10 % w / v casamino acids , 5 % w / v glycerol , 0 . 5 mg / ml ampicillin ( mcga media ) at 30 ° c . for 24 hrs with aeration ( 250 rpm ). 30 - 50 ml of this culture was used to inoculate 500 ml of the same mcga media and grown for 12 - 16 hrs overnight at 30 ° c . with aeration ( 250 rpm ) and then iptg added to a final concentration of 0 . 2 - 0 . 5 mm . cells were induced for 12 - 24 hrs by growth at 30 ° c . with aeration ( 250 rpm ) and then stood on ice for 10 min . cells were pelleted by centrifugation for 30 min , 10 , 000 rpm , followed by 30 min at 14 , 000 rpm and the supernatant filtered through a 0 . 45 μm filter unit ( nalgene ). the supernatant was concentrated 10 - 20 fold in a high pressure concentrator with a ym10 filter and then dialyzed overnight ( 3 changes ) against pbs at 4 ° c . the dialyzed supernatant was passed through a ni 2 + - nta - agarose column using the procedure for isolation from e . coli host according to example 2 . alternatively , the s . marcescens recombinants may be induced for shorter time periods and the protein isolated from the periplasmic space by osmotic shocking . yields are higher if longer induction periods are employed and the protein isolated from culture supernatant . the folded state of the recombinant tcr fragments was assessed by circular dichroism ( cd ) analysis . results indicated significant proportion of β - sheet structure , strongly suggesting native folding . [ 0121 ] fig1 shows the circular dichroism spectra of recombinant tcr proteins . the recombinant tcr fragments were purified using the methodology described above , from e . coli cells harboring v α pelbhis , v β pelbhis and scv α v β pelbhis and dialyzed into 10 mm sodium phosphate ph7 . 0 prior to cd analysis . as a comparison , the immunoglobulin scfv fragment derived from the d1 . 3 antibody ( ward et al ., 1989 ) was purified and used . this fragment was expressed from a plasmid construction derivative of psw2 ( ward et al ., 1989 ; mccafferty et al ., 1990 ). the scfv was purified from the culture supernatant of induced cultures using lysozyme sepharose ( ward et al ., 1989 ) and dialyzed against 10 mm sodium phosphate ph 7 . 0 prior to analysis in cd . the rationale for using this immunoglobulin fragment as a comparison is that molecular modeling indicates that the v α and v β domains of tcrs resemble immunoglobulin variable domains ( chethi et al ., 1988 ; novotny et al ., 1986 ). for each recombinant tcr protein , several spectra were generated using different concentrations and / or protein from different purification batches . fig5 shows representative spectra . cd analyses were carried out using an aviv model 60ds circular dichroism spectrophotometer at 25 ° c . and a cell path of 0 . 2 cm . concentrations of proteins in 10 mm nah 2 po 4 varied from 1 . 0 μm to 7 . 8 μm . concentration of the purified proteins was determined by quantitative amino acid hydrolysis . proteins examined were v α ( his ) 6 , v β ( his ) 6 , sctcrv α v β ( his ) 6 and d1 . 3scfv . by comparison with both the cd spectrum of the structurally solved d1 . 3 fv fragment ( bhat et al ., 1990 ) and that of other proteins known to have a high proportion of β - pleated sheet structure , it was concluded that the cd spectra of the recombinant tcr fragments contained a high proportion of β - pleated sheet structure . this is consistent with the molecular modeling studies which indicate that the tcr domain fold is immunoglobulin - like in character ( chothia et al ., 1988 ; 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