BACKGROUND
the antigen  specific receptor of the t cell lineage, the tcr, is a transmembrane heterodimer of covalently coupled α- and β-polypeptide chains.
each chain consists of two extracellular immunoglobulin  domains, one variable  and one constant , and the two v domains comprise the ligand binding portion that specifically interacts with a peptide/major histocompatibility complex .
tcrs are detection molecules with exquisite specificity and exhibit, like abs, an enormous diversity.
the fine tuning of the specificity, mhc restriction and thymic selection is incompletely understood.
however, recent re-evaluation and comparison of the existing pmhc/tcr crystal structures demonstrate conservation of specific tcr-mhc contacts in complexes bearing common v segments and mhc allotypes.
this has for the first time made it possible to postulate a tcr-mhc recognition code  <cit> .
however, the predictions are still based on a restricted number of crystallographic data sets primarily due to the lack of a robust and versatile expression system for soluble tcrs necessary for obtaining sufficient amounts of protein.
soluble tcrs are prone to aggregation and missfolding, and many of the difficulties encountered may probably be explained by the fact that tcrs, unlike abs, have not evolved to be secreted, but are expressed as membrane-bound molecules that are intrinsically unstable when expressed as soluble molecules.
several engineering strategies for making soluble versions of tcrs, including sctcrs  <cit>  and fusion of the extracellular tcr domains to other proteins; i.e.
maltose binding protein, human constrant kappa domain  or leucine zippers  <cit>  have been reported.
however, all of these strategies have shown limited success due to low production yield, poor functionality, or lack of crystallization abilities  <cit> .
the introduction of a non-native disulphide bond in the tcr invariant region, to make so called dstcr, has greatly increased the stability and folding characteristics of more than  <dig> human tcrs when expressed as cytosolic inclusion bodies that have been refolded  <cit> .
even without this artificial disulphide bond, optimized bacterial inclusion body expression and refolding has so far shown the highest success rate for obtaining soluble tcrs in high yields  <cit> .
recently an improved strategy for soluble periplasmic e. coli expression based on rational mutagenesis, over-expression of skp, and fusion to the ab cκ domain, was reported  <cit> .
however, all of the expression systems described so far represents labour intensive and low through-put avenues that require either inclusion body denaturation and refolding  <cit> , introduction of solubility-increasing amino acid substitutions or fusion to a second protein which might interfere with downstream applications  <cit> .
the extracellular part of tcrs and ab fab fragments are structurally similar.
this is also the case for molecules that consist of the two v domains connected by a flexible linker, namely single chain fragment variable  and sctcrs, respectively.
e. coli expression of ab derived fragments has been highly successful and a number of vector systems and expression strategies exist .
these are to a large extent based on direct targeting to the periplasm with or without co-expression of chaperones, such as skp, fkp, trigger factor and dsbc  <cit> .
in the current report, we describe an improved periplasmic expression system that allows rapid expression of unmodified soluble sctcrs based on over-expression of fkpa.
the expression system was developed in e. coli xl1-blue to complement a recently reported tcr phage display platform based on this particular host  <cit> .
a systematic evaluation of a variety of parameters known to influence heterologous protein expression in e. coli was conducted using two sctcrs derived from the t cell clones 4b2a <dig> and 7a10b <dig> associated with a mouse model for idiotype  dependent immune regulation  <cit> .
notably, these sctcrs are genetically unrelated.
the final expression and purification protocol allowed the isolation of monomeric soluble sctcrs, with correct native fold and apparent functional activity.
key advantages over earlier tcr expression systems are the ability to produce "native" tcr sequences without previous engineering steps such as the introduction of solubility increasing mutations or fusion proteins and the ability to generate soluble material without refolding strategies.
this system can therefore be used as a soluble expression platform downstream of a phage display library selection, or as an initial screen of a panel of native tcr clones to pick out the best starting point before stability or affinity maturation engineering.
methods
abs and additional reagents
the mouse anti-his-hrp ab was purchased from abd serotec .
the anti-flag m2-hrp ab was purchased from sigma-aldrich .
all restriction enzymes were purchased from new england biolabs .
tcr-specific mabs were gb <dig>  <cit>  , f <dig>   <cit>  , 44-22- <dig>  <cit>  , rr4- <dig>  <cit>  .
f <dig>  and 44-22- <dig> were kind gifts from dr. uwe d. staerz  and dr. hans hengartner , respectively.
rr4- <dig> was purchased from bd pharmingen .
the scfv control was a kind gift form affitech as .
t cell clones
4b2a <dig> and 7a10b <dig> t cell clones are specific for residues 91- <dig> of mouse ig λ <dig> light chain presented on the mhc class ii i-ed molecule  <cit> .
the clones are distinguished by the 4b2a <dig> not cross-reacting to a phe <dig>  to tyr <dig>  substitution while 7a10b <dig> does  <cit> .
moreover, while 4b2a <dig> expresses a unique tcr , 7a10b <dig> expresses a dominant and recurrent tcr  in this system  <cit> .
plasmids and bacterial strains
the soluble expression vectors phog-dummy  <cit>  and phog <dig>  <cit>  were a kind gift from affitech as .
phog <dig> encodes a scfv with specificity against 2-phenyloxazol-5-one  coupled to bovine serum albumin , originally isolated from a human ab phage library  <cit> .
the pfkpdn phagemid encodes either the 4b2a <dig>  or 7a10b <dig>  sctcrs that have been described in detail previously  <cit> .
e. coli xl- <dig> blue ] was purchased from stratagene .
construction of the sctcr phog, pfkpen and pfkpei expression vectors
the phog expression vectors  were generated by subcloning the sctcr encoding cassettes from pfkpdn phagmid into phog on compatible ncoi/noti re sites using standard techniques, before transformation into e. coli xl1-blue cells.
likewise, the pfkpen expression vectors  were created by subcloning the fkpa expression cassette from pfkpdn into these new vectors on compatible scai/nhei re sites.
the fkpa gene is constitutively expressed from its native promoter, xpo, in pfkpen .
to generate an expression vector were fkpa transcription is controlled by the lac promoter that also controls the sctcr transcription, the pfkpei expression vector was made .
the genomic e. coli k <dig> sequence  was used for primer design to isolate the fkpa gene orf.
the forward primer was designed with a 5'-end nhei tag followed by the t7g <dig> tir region  <cit>  and the fkpa-encoding portion .
the reverse primer also included a 5'-end nheli re site and a flag tag encoding sequence .
pcr was applied to  <dig> μl of e. coli xl1-blue overnight culture using vent dna polymerase .
the resulting dna fragment was ligated into phog-dummy  <cit>  on the corresponding nhei re site creating the new pfkpei-dummy expression vector.
to generate the final pfkpei expression vector , the ncoi/noti sctcr segments were cloned into this construct as described above, before transformation into e. coli xl1-blue cells.
the pfkpei expression vector  contains a dicistronic expression cassette with the sctcr open reading frame  followed by a translation initiation region  and the fkpa orf, both controlled by the lac promoter.
all the constructs were validated by re analysis and subsequently confirmed by sequencing.
protein production
e. coli xl1-blue cells transformed with the plasmids were inoculated from glycerol stocks into  <dig> ml lb-medium supplemented with  <dig> μg/ml tetracycline,  <dig> μg/ml ampicillin and  <dig>  m glucose , in  <dig> ml tubes and incubated at 37°c/220-rpm overnight .
the cultures were then re-inoculated into  <dig> ml lbtag medium at an od <dig> nm of  <dig>  in  <dig> ml tubes and incubated at 37°c/ <dig> rpm.
at od <dig> nm  <dig> - <dig> , the bacteria were pelleted  and the pellets resuspended in  <dig> ml lb medium supplemented with  <dig>  mm isopropyl-β-d-thio-galactopyranoside ,  <dig> μg/ml tetracycline and  <dig> μg/ml ampicillin.
cultures were then incubated at 30°c/ <dig> rpm/on.
to directly compare specific sctcr and fkpa protein production from each clone, cell numbers were normalized according to od <dig> nm before being channelled into an identical cellular compartment fractionation procedure.
the standard expression conditions described above were varied such that the cultures were grown under standard conditions until od <dig> nm  <dig> - <dig> , pelleted and resuspended in lb-medium supplemented with the following additives, either  <dig>  mm iptg and  <dig>  m sucrose,  <dig>  m sucrose or without any additives.
again, cell numbers were normalized as above.
an up-scaled shaker flask production was performed in  <dig> ml expression cultures without any additives for 4b2a <dig> sctcr vαβ in pfkpei, while 4b2a <dig> sctcr vβα in pfkpei was performed with  <dig>  mm iptg additive, in  <dig> l shaker flasks.
7a10b <dig> sctcr vαβ and vβα in pfkpen was produced in several small scale cultures  without any additives.
all cultures were then subject to the cellular compartment fractionation procedure.
cellular compartment fractionation
cells were harvested by centrifugation at  <dig> min/4600-g/4°c and separated into;  medium fraction,  soluble periplasmic fraction and  cytosolic fraction by a modified version of that described by kipriyanov et al.
<cit> .
briefly, the medium fraction was obtained by an additional centrifugation of  <dig> ml supernatant .
to obtain the cytosolic and periplasmic fractions, the pellet was resuspended in  <dig> μl ice-cold periplasmic extraction solution  supplemented with  <dig> μl lysozyme  and  <dig> μl rnase a  before incubation for  <dig> h at 4°c with rotation.
the mixture was centrifuged, leaving the soluble periplasmic fraction as the supernatant.
the pellet, containing the cytosolic fraction, was resuspended in  <dig> μl ice-cold  <dig>  m tris-hcl, ph  <dig>  before addition of  <dig> μl 4× sample buffer  and subjected to rough pipetting to dissolve the pellet.
for the large scale  cultures, the same fractionation procedure was used after adjusting the reagent volumes ×  <dig>
protein purification
the periplasmic fractions isolated after 400-ml expression were diluted 1: <dig> in binding buffer  and filtered through a  <dig>  μm sterile filter .
the samples were purified by immobilized metal affinity chromatography  on a his-traphp column  as described in the protocol provided by the manufacturer.
the eluted protein fractions were pooled, concentrated by amicon ultra- <dig> centrifugal filter units  and dialyzed against storage buffer .
protein concentration was determined by a <dig> nm on a nanodrop nd- <dig> apparatus  based on calculated extinction coefficients  <cit> .
size exclusion chromatography  was performed on an automated Δkta chromatography system,  using  <dig> μl of the imac purified protein to isolate monomeric sctcr.
the concentration of protein in each fraction was determined as described above, followed by analysis on non-reducing and reducing sds-page and western blotting as described below.
the monomeric fractions were pooled, concentrated by amicon ultra- <dig> centrifugal filter units  followed by concentration determination as before.
sds-page and western blotting
identical volumes of fractionated samples isolated in parallel from a <dig> nm normalized e. coli cell cultures were separated by non-reducing sds-page on 4-12% bis-tris xt precast gels  and blotted onto a polyvinylidene fluoride membrane  in tris/glycine buffer  at  <dig> v for  <dig> min using a semi-dry blotting apparatus .
the membrane was blocked in pbstm  before sctcr proteins were detected with anti his-hrp ab .
the membrane was washed and developed with supersignal™ west pico substrate  before exposed to biomax mr film .
expression of rfkpa was detected after  <dig> min stripping by restore™ western blot stripping buffer , and the membrane redeveloped as above using an anti-flag m2-hrp ab .
samples of  <dig>  μg superdex  <dig> purified sctcrs were analyzed in parallel with  <dig>  μg imac purified sctcrs by non-reducing and reducing sds-page followed by western blotting as described above.
sctcr capture elisa
the various mabs were absorbed to maxisorp microtiter plate wells  at a concentration of  <dig> μg/ml in  <dig> × pbs, ph  <dig>  on at 4°c.
the wells were blocked with pbstm for  <dig> h at rt, and imac purified sctcr samples at a concentration of  <dig> μg/well and superdex <dig> purified sctcr samples at a concentration of  <dig>  μg/well were added.
all were allowed to react for  <dig> h at rt before the captured sctcrs were detected with anti-his-hrp ab  for  <dig> h at rt.
the wells were developed with  <dig> μl of tmb soluble substrate , stopped by adding  <dig> m hcl after  <dig> min, and the absorbance read at a <dig> nm.
circular dichroism  spectroscopy
cd spectra were recorded using a jasco j- <dig> spectropolarimeter .
measurements were performed at 25°c using a quartz cuvette  with a path length of  <dig>  cm.
measurements were performed with a protein concentration of  <dig>  mg/ml for 4b2a <dig> sctcr vαβ and  <dig>  mg/ml for the control scfv ab fragment in 1× pbs, supplemented with  <dig> mm nacl .
samples were scanned  <dig> times at  <dig> nm/min, using a band width of  <dig> nm.
the response time was  <dig> s, and the wave length range was 200- <dig> nm.
the data were averaged and the spectrum of a sample-free control was subtracted.
the secondary structure elements of the proteins were calculated from ellipticity data, using the spectral fitting methods, contin/ll  <cit> , selcon <dig>  <cit>  and cdsstr  <cit> , in the cdpro package  <cit> .
thermal denaturation was determined as the change in cd signal from heating.
the temperature was controlled with a tpc-423s/l system .
secondary structure prediction
secondary structure element of the 2c tcr structure   <cit>  were predicted using the dssp  <cit>  and stride <cit> .
RESULTS
construct design
the phog , pfkpen  and pfkpei  vectors drive sctcr expression from the lac promoter.
in all cases, the sctcrs are fused to an n -terminal pelb signal sequence that directs protein translocation across the inner membrane of e. coli through the secb-dependent pathway to the oxidising environment of the periplasm.
this oxidising environment is a prerequisite for generation of disulphide bridges within proteins such as sctcrs  <cit> .
the pfkpen and pfkpei vectors also carry the gene encoding the periplasmic chaperone fkpa.
in pfkpen, recombinant fkpa  expression is controlled by its native promoter, while pfkpei contains a dicistronic expression cassette where rfkpa gene transcription is controlled by the lac promoter.
in all vectors, the sctcr cassette was constructed by genetically fusing vα and vβ encoding gene segments with a sequence encoding a synthetic linker  <cit> .
to gain insight into how domain orientation affects soluble expression, two versions with alternative domain orientation were made, such that either vα  or vβ  was expressed n-terminally.
all sctcrs were fused to a c-myc- and a his-tag c-terminally, whilst rfkpa was fused to a c-terminal flag-tag.
all constructs used in the current study are described in figure  <dig>
expression level and subcellular localization
expression was performed in e. coli xl1-blue followed by cell fractionation and periplasmic expression analyzed by sds-page and western blotting, as described in the method section.
as shown in figure  <dig>  4b2a <dig> sctcr was not detected in the periplasmic fraction using anti-tag mabs in the absence of rfkpa over-expression , regardless of domain orientation and expression conditions.
the 7a10b <dig> sctcr behaved like 4a10b <dig> .
thus, both sctcrs failed to reach the periplasmic space, and rather aggregated in the cytosol .
the growth characteristics of each culture were studied and the results are summarized in table  <dig>  the one expressing 4b2a <dig> vαβ sctcr showed sign of growth arrest.
all others demonstrated overall robust growth and reached a final cell density similar to that of the xl1-blue control without plasmid.
a a  <dig> nm after on incubation of  <dig> ml expression cultures
b no growth of cultures after induction
the chaperone fkpa has a highly beneficial effect on periplasmic expression and correct folding of toxic ab scfvs,  <cit> , and we have recently reported a similar effect on phage display of sctcrs  <cit> .
thus, the effect of fkpa over-expression was assessed here.
the clones that were picked for further study, all expressed high levels of fkpa as demonstrated by sds-page and western blot of whole cell lysates using anti-flag ab .
the periplasmic levels of sctcrs in both configurations, vαβ and vβα, were detected under different expression conditions.
the levels of rfkpa in the periplasm were also assessed.
when expression was induced with iptg, a high yield of 4b2a <dig> sctcr in vβα orientation was observed, while sctcr with the opposite vαβ orientation was not .
the highest yield of 4b2a <dig> vβα sctcr was obtained when rfkpa transcription was under lac promoter control.
all four of these 4b2a <dig> sctcr producing cultures  showed robust growth .
the 7a10b <dig> sctcr was not detected or detected in negligible amounts in the periplasmic fraction regardless of orientation .
the sctcr vαβ producing culture showed sign of growth arrest when rfkpa was transcribed from its endogenous promoter .
it has earlier been reported that a reduction of the iptg concentration has a positive effect on periplasmic yield of soluble protein expressed from a lac promoter  <cit> .
indeed, when expression was performed without iptg, the periplasmic yield of 4b2a <dig> vαβ increased considerably.
the yield of 4b2a <dig> vβα, on the other hand, decreased .
the yield of 7a10b <dig> sctcr in both domain orientations increased .
all cultures reached high and similar final cell density .
analysing periplasmic fkpa levels, this was equal to or higher than that observed in the presence of iptg in all cases, except when expressed from the lac promoter in the vector encoding 4b2a <dig> sctcr vβα.
this was in line with the fact that very little 4b2a <dig> sctcr vβα was found in the periplasmic space in the absence of induction.
addition of  <dig>  m sucrose to the growth medium has been shown to increase the yield of secreted proteins, either in the periplasm or culture medium  <cit> .
to investigate if the soluble sctcr yield in the periplasmic fraction could be further increased, the effect of sucrose addition was assessed both in uninduced and iptg induced cultures.
neither the 4b2a <dig>  nor 7a10b <dig>  sctcr levels increased after sucrose addition.
the expression patterns were identical to that observed without sucrose addition, and no increased leakage to the culture medium was observed .
taken together, overall good yields of periplasmic 4b2a <dig> sctcr were seen in the presence of periplasmic rfkpa expression.
this was observed from both pfkpen and pfkpei plasmids; for vαβ, when expression was performed in the absence of iptg and for vβα, when expression was performed in the presence of iptg.
for 7a10b <dig> sctcr, the overall highest yield was seen from the pfkpen plasmid in the absence of iptg for both the vαβ and the vβα configuration.
protein purification
the four sctcr variants, 4b2a <dig> and 7a10b <dig> in both domain orientations, were expressed at large scale under conditions shown to give good small scale periplasmic yields.
protein fractionation was performed as described above, followed by imac purification.
eluted fractions containing protein were pooled and concentrated.
the production yields after concentration is given in table  <dig> and were  <dig>  and  <dig>  mg/l for the 4b2a <dig> vαβ and vβα variants, respectively.
very low yield of the 7a10b <dig> variants were obtained.
thus, the two 4b2a <dig> samples only were further analyzed by size exclusion chromatography  , followed by non-reducing and reducing sds-page of eluted fractions .
importantly, the vαβ format had more than double the amount of monomeric sctcr compared to the vβα format .
however, both formats were produced as polymers that appeared to be composed of both non-covalent and covalent aggregates .
taken together, the overall best yield of monomeric sctcr was from the 4b2a <dig> clone in the vαβ orientation.
this format yielded  <dig>  mg per l culture of active purified monomeric material, which is comparable with or better than amounts acquired by others using periplasmic expression systems   <cit> .
importantly, the hucκ fusion format reported by maynard et al  <cit>  requires downstream proteolytically removal of the hucκ moiety, hence the actual soluble sctcr yield will be equal to or lower than with our approach.
moreover, the absence of this hucκ fusion resulted in wastely reduced sctcr yield   <cit> .
a estimated periplasmic yield of total soluble sctcrs after imac purification.
b estimated periplasmic yield of monomeric soluble sctcr after size exclusion chromatography.
c values implemented from maynard et al  <cit> , sctcr-human c kappa fusion format
d values implemented from maynard et al  <cit> , sctcr format
nd; not determined
folding and secondary structure analysis
elisa
the conformational integrity of the imac and size exclusion chromatography purified 4b2a <dig> sctcrs was analyzed using two v-gene specific mabs in a capture elisa.
furthermore, 7a10b <dig> sctcrs were produced in several parallel small scale cultures, that were pooled, imac purified and analyzed in capture elisa.
the results show that differences exist in mab reactivity between the sctcrs with regard to domain orientation.
for both 4b2a <dig> and 7a10b <dig> sctcr, the vαβ sctcrs exhibited stronger mab reacitivity than the vβα counterparts .
both anti-vβ <dig> mab f <dig>  and clonotype-specific gb <dig> bound the purified 4b2a <dig> sctcrs fractions .
gb <dig> bound the two molecules equally well, whilst f <dig>  bound better to vαβ than to vβα, likewise, the imac purified 7a10b <dig> vαβ sctcr was detected by the vβ6-specific 44-2- <dig> and rr4- <dig> mabs, whereas no binding was observed in the vβα domain orientation .
structural analysis by cd spectroscopy
sctcr vαβ 4b2a <dig> and a control scfv ab fragment were subjected to cd spectroscopy at 20°c and 56°c as shown in figure  <dig>  and the spectra obtained analyzed using contin/ll  <cit> , selcon <dig>  <cit>  and cdsstr  <cit> .
the obtained structure data from the three fitting techniques were similar.
however, contin/ll gave slightly lower rmsd ratios than cdsstr.
the results from contin/ll are thus given in table  <dig>  the cd data analysis indicated a 47% and 42% β-sheet content as well as a 5% and 5% α-helix content in the scfv control and 4b2a <dig> sctcr vαβ molecules, respectively.
the spectra of both 4b2a <dig> sctcr vαβ  and the control scfv ab fragment  are hence typical of a protein with high degree of β-sheet structure characteristic for the ig-fold.
this is consistent with the secondary structure element prediction given by dssp  <cit>  and stride  <cit>  for the 2c tcr structure   <cit> , which calculates a 49% β-sheet content and a α-helix content of  <dig> %.
the degree of secondary structure content decreased as the temperature was raised towards 56°c as seen in table  <dig> and figure 6c, indicating thermally induced unfolding of the protein.
a the secondary structure elements were estimated by contin/ll from the cd spectrums presented in figure  <dig>  the experiment was repeated twice, and representative results are shown.
discussion
the aim of the current study was to generate a general expression system for soluble sctcrs.
the 4b2a <dig> and 7a10b <dig> sctcrs used have previously been successfully expressed as fusions to piii on filamentous phage  <cit> .
based on that study, the current report focuses on the soluble sctcr format and investigation of protein yield and sub-cellular localization using a bacterial system compatible with the e. coli host used in sctcr phage display, namely xl1-blue.
chaperones are known to enhance expression yields as they facilitate folding, prevent aggregation, reactivate aggregates and reduce protein degradation  <cit> .
bothmann and plückthun  <cit>  identified two periplasmic factors, namely fkpa and skp, which increased the functional periplasmic yields of toxic ab scfvs, while already well-expressing clones were unaffected.
fkpa is a periplasmic peptidyl-propyl cis,trans-isomerase, but the effect was also striking for scfvs that did not contain cis-prolines.
the positive effect rather seemed to be a result of the ability of the chaperone to prevent premature aggregation of early folding intermediates as well as reactivation of inactive proteins  <cit> .
skp belongs to a family of cavity-containing chaperons and works by protecting its substrate from aggregation within this cavity  <cit> .
skp was recently reported to have an effect on sctcr expression  <cit> .
however, co-expression of skp and fkpa showed no synergistic effect on scfv yield.
rather, co-expression had the same effect as fkpa alone  <cit> .
we have previously reported that over-expression of rfkpa normalized the display level of sctcrs-piii fusions in phage display, while skp had only minor effects  <cit> .
in the present study, we therefore focused on fkpa co-expression.
without chaperone co-expression, the sctcrs were trapped in the cytosol despite the fact that all constructs were equipped with a pelb leader sequence directing transport to the periplasm.
when fkpa was co-expressed, we observed a positive effect on growth rate.
moreover, a profound effect on the accumulation of soluble 4b2a <dig> and 7a10b <dig> sctcrs in the periplasm was also seen.
this was the case whether rfkpa was expressed from its native promoter or the lac promoter.
these initial results hence showed that fkpa has an ability to significantly increase the periplasmic expression yield of the tcrs.
the effect of domain orientation on functional sctcr fragments has been reported earlier, and show the same trend as we see here, namely that the vα-linker-vβ domain orientation increases the functional fraction of sctcr fragments  <cit> .
generally, the most unstable fragment should be put n-terminally and the most stable fragment c-terminally.
this prevents rapid folding of the n-terminal domain followed by abortive periplasmic translocation caused by a partly unfolded c-terminal domain  <cit> .
the trbv13- <dig> segment used in the 4b2a <dig> sctcr, is considered a particularly stable fragment  <cit> .
the results in this report hence fit well with the results obtained by others.
culture conditions, such as induction and growth in the presence of certain sugars, have been shown to influence the production yield and sub-cellular localization of proteins  <cit> .
we therefore investigated the effect of sucrose addition to the medium as well as the effect of adding or removing iptg.
the results showed that 4b2a <dig> tcr in the vβα orientation was expressed well under standard conditions, that is, in the presence of iptg, but without addition of sugars.
in contrast, 4b2a <dig> tcr vαβ and 7a10b <dig> in both domain orientations were expressed poorly.
removal of iptg from the growth medium restored the expression of these three molecules.
without iptg residual transcription still occurs through the leaky lac promoter, but with decreased translation and hence folding load.
this was found to be beneficial for the sctcrs.
it has previously been reported that growing cells in the presence of certain sugars, raffinose or sucrose, can reduce the aggregation of recombinant proteins in the e. coliperiplasm  <cit> .
these sugars are small enough to diffuse through the inner membrane to the periplasm, causing an increase in the osmotic pressure resulting in enlargement of the periplasmic space, and hence a decrease in the local protein concentration  <cit> .
however, the addition of sucrose had no effect on the yield of sctcr in the periplasm.
possibly, the over-expression of rfkpa prevents premature aggregation so efficiently that the addition of sucrose has little or no detectable effect.
when large scale expression using optimalized expression conditions for each of the sctcrs was performed mg/l amounts were obtained from both 4b2a <dig> sctcrs after purification.
the 7a10b <dig> derived sctcrs showed an altered expression profil after up-scaling, which has also been reported by others for antibody scfv fragments  <cit> .
however, by pooling the periplasmic fraction from several small scale cultures, yields of μg/l were obtained.
thus, the 4b2a <dig> derived molecules only were chosen for studies of how domain orientation influences the yield of the monomeric fraction as well as folding.
we found that the sctcr with the vαβ domain orientation had a larger monomeric peek than the one with the vβα domain orientation.
aggregates appeared to be formed after misfolding due to erroneous disulphide bond formation as well as through surface exposed hydrophobic patches.
the affinity of the monomers for their cognate pmhc  ligand, is too low for detection by flow cytometry.
we therefore used binding to several well characterized mabs as an indirect measurement of structural integrity and functionality.
the same mabs have been used earlier to characterize the same sctcrs displayed on phage  <cit>  or expressed as tcr-ig fusions .
in both cases, binding to mabs correlated with the ability of the tcr moiety to bind i-ed expressing murine a <dig> lymphoma cells that had been loaded with the cognate λ <dig> peptide, and binding required a multivalent format   <cit> .
importantly, the current report shows differences in mab reactivity with regard to domain orientation, and the vαβ format was superior to the vβα format.
furthermore, the secondary structure data given by cd measurements shows that the sctcr vαβ has a native-like fold that in conjunction with the binding activity of the mabs strongly indicates functionality.
CONCLUSIONS
the work demonstrated that the presence of the chaperone fkpa, expression conditions as well as domain orientation greatly influence the yield and sub-cellular localization of soluble tcrs.
over-expression of fkpa in the periplasm is a prerequisite for soluble periplasmic localization of the sctcr, and absence of inducer  further improved the periplasmic yield of formats which were poorly expressed under standard conditions.
addition of sucrose to the medium did not influence the yield.
comparing sctcr molecular design, the format with vαβ domain orientation gives a better expression profile with regard to monomeric protein yield than the vβα format, and the binding to specific mabs is stronger.
furthermore, the purified 4b2a <dig> sctcr vαβ exhibited well resolved secondary structure elements typical for the ig fold topology indicating a correctly folded tcr moiety.
the final protein yields obtained using our optimized approach perform equal or better than previously reported periplasmic expression systems and this was achieved without fusions or aa modifications of the tcr moiety.
we anticipate that this system may enable rapid production of a wide range of soluble tcrs, and thereby simplify the screening of a new generation of recombinant tcrs generated by protein engineering and display techniques such as phage-display.
authors' contributions
ksg did most of the experimental work, participated in the design of the study and drafted the manuscript.
el gave initial technical guidance.
pek gave guidance on the cd analysis.
bb provided key reagents, participated in the design of the study and in drafting the manuscript.
gÅl designed and constructed the expression vectors, supervised the study and participated in drafting and finalizing the manuscript.
is supervised the study, provided funding and finalized the manuscript.
all authors approved the final manuscript.
supplementary material
additional file 1
primer overview.
primers used to isolate the fkpa gene orf.
click here for file
acknowledgements
the authors would like to thank sathiyaruby manicam vladivelu for excellent technical assistance.
this work was supported by the norwegian research council .

 the antigen specific receptor of the t cell lineage, the tcr, is a transmembrane heterodimer of covalently coupled α- and β-polypeptide chains. each chain consists of two extracellular immunoglobulin domains, one variable and one constant, and the two v domains comprise the ligand binding portion that specifically interacts with a peptide/major histocompatibility complex. tcrs are detection molecules with exquisite specificity and exhibit, like abs, an enormous diversity. the fine tuning of the specificity, mhc restriction and thymic selection is incompletely understood. however, recent re-evaluation and comparison of the existing pmhc/tcr crystal structures demonstrate conservation of specific tcr-mhc contacts in complexes bearing common v segments and mhc allotypes. this has for the first time made it possible to postulate a tcr-mhc recognition code. however, the predictions are still based on a restricted number of crystallographic data sets primarily due to the lack of a robust and versatile expression system for soluble tcrs necessary for obtaining sufficient amounts of protein. soluble tcrs are prone to aggregation and missfolding, and many of the difficulties encountered may probably be explained by the fact that tcrs, unlike abs, have not evolved to be secreted, but are expressed as membrane-bound molecules that are intrinsically unstable when expressed as soluble molecules. several engineering strategies for making soluble versions of tcrs, including sctcrs and fusion of the extracellular tcr domains to other proteins; i.e. maltose binding protein, human constrant kappa domain or leucine zippers have been reported. however, all of these strategies have shown limited success due to low production yield, poor functionality, or lack of crystallization abilities. the introduction of a non-native disulphide bond in the tcr invariant region, to make so called dstcr, has greatly increased the stability and folding characteristics of more than human tcrs when expressed as cytosolic inclusion bodies that have been refolded. even without this artificial disulphide bond, optimized bacterial inclusion body expression and refolding has so far shown the highest success rate for obtaining soluble tcrs in high yields. recently an improved strategy for soluble periplasmic e. coli expression based on rational mutagenesis, over-expression of skp, and fusion to the ab cκ domain, was reported. however, all of the expression systems described so far represents labour intensive and low through-put avenues that require either inclusion body denaturation and refolding, introduction of solubility-increasing amino acid substitutions or fusion to a second protein which might interfere with downstream applications. the extracellular part of tcrs and ab fab fragments are structurally similar. this is also the case for molecules that consist of the two v domains connected by a flexible linker, namely single chain fragment variable and sctcrs, respectively. e. coli expression of ab derived fragments has been highly successful and a number of vector systems and expression strategies exist. these are to a large extent based on direct targeting to the periplasm with or without co-expression of chaperones, such as skp, fkp, trigger factor and dsbc. in the current report, we describe an improved periplasmic expression system that allows rapid expression of unmodified soluble sctcrs based on over-expression of fkpa. the expression system was developed in e. coli xl1-blue to complement a recently reported tcr phage display platform based on this particular host. a systematic evaluation of a variety of parameters known to influence heterologous protein expression in e. coli was conducted using two sctcrs derived from the t cell clones 4b2a and 7a10b associated with a mouse model for idiotype dependent immune regulation. notably, these sctcrs are genetically unrelated. the final expression and purification protocol allowed the isolation of monomeric soluble sctcrs, with correct native fold and apparent functional activity. key advantages over earlier tcr expression systems are the ability to produce "native" tcr sequences without previous engineering steps such as the introduction of solubility increasing mutations or fusion proteins and the ability to generate soluble material without refolding strategies. this system can therefore be used as a soluble expression platform downstream of a phage display library selection, or as an initial screen of a panel of native tcr clones to pick out the best starting point before stability or affinity maturation engineering. methods abs and additional reagents the mouse anti-his-hrp ab was purchased from abd serotec. the anti-flag m2-hrp ab was purchased from sigma-aldrich. all restriction enzymes were purchased from new england biolabs. tcr-specific mabs were gb, f, 44-22-, rr4-. f and 44-22- were kind gifts from dr. uwe d. staerz and dr. hans hengartner, respectively. rr4- was purchased from bd pharmingen. the scfv control was a kind gift form affitech as. t cell clones 4b2a and 7a10b t cell clones are specific for residues 91- of mouse ig λ light chain presented on the mhc class ii i-ed molecule. the clones are distinguished by the 4b2a not cross-reacting to a phe to tyr substitution while 7a10b does. moreover, while 4b2a expresses a unique tcr, 7a10b expresses a dominant and recurrent tcr in this system. plasmids and bacterial strains the soluble expression vectors phog-dummy and phog were a kind gift from affitech as. phog encodes a scfv with specificity against 2-phenyloxazol-5-one coupled to bovine serum albumin, originally isolated from a human ab phage library. the pfkpdn phagemid encodes either the 4b2a or 7a10b sctcrs that have been described in detail previously. e. coli xl- blue ] was purchased from stratagene. construction of the sctcr phog, pfkpen and pfkpei expression vectors the phog expression vectors were generated by subcloning the sctcr encoding cassettes from pfkpdn phagmid into phog on compatible ncoi/noti re sites using standard techniques, before transformation into e. coli xl1-blue cells. likewise, the pfkpen expression vectors were created by subcloning the fkpa expression cassette from pfkpdn into these new vectors on compatible scai/nhei re sites. the fkpa gene is constitutively expressed from its native promoter, xpo, in pfkpen. to generate an expression vector were fkpa transcription is controlled by the lac promoter that also controls the sctcr transcription, the pfkpei expression vector was made. the genomic e. coli k sequence was used for primer design to isolate the fkpa gene orf. the forward primer was designed with a 5'-end nhei tag followed by the t7g tir region and the fkpa-encoding portion. the reverse primer also included a 5'-end nheli re site and a flag tag encoding sequence. pcr was applied to μl of e. coli xl1-blue overnight culture using vent dna polymerase. the resulting dna fragment was ligated into phog-dummy on the corresponding nhei re site creating the new pfkpei-dummy expression vector. to generate the final pfkpei expression vector, the ncoi/noti sctcr segments were cloned into this construct as described above, before transformation into e. coli xl1-blue cells. the pfkpei expression vector contains a dicistronic expression cassette with the sctcr open reading frame followed by a translation initiation region and the fkpa orf, both controlled by the lac promoter. all the constructs were validated by re analysis and subsequently confirmed by sequencing. protein production e. coli xl1-blue cells transformed with the plasmids were inoculated from glycerol stocks into ml lb-medium supplemented with μg/ml tetracycline, μg/ml ampicillin and m glucose, in ml tubes and incubated at 37°c/220-rpm overnight. the cultures were then re-inoculated into ml lbtag medium at an od nm of in ml tubes and incubated at 37°c/ rpm. at od nm -, the bacteria were pelleted and the pellets resuspended in ml lb medium supplemented with mm isopropyl-β-d-thio-galactopyranoside, μg/ml tetracycline and μg/ml ampicillin. cultures were then incubated at 30°c/ rpm/on. to directly compare specific sctcr and fkpa protein production from each clone, cell numbers were normalized according to od nm before being channelled into an identical cellular compartment fractionation procedure. the standard expression conditions described above were varied such that the cultures were grown under standard conditions until od nm -, pelleted and resuspended in lb-medium supplemented with the following additives, either mm iptg and m sucrose, m sucrose or without any additives. again, cell numbers were normalized as above. an up-scaled shaker flask production was performed in ml expression cultures without any additives for 4b2a sctcr vαβ in pfkpei, while 4b2a sctcr vβα in pfkpei was performed with mm iptg additive, in l shaker flasks. 7a10b sctcr vαβ and vβα in pfkpen was produced in several small scale cultures without any additives. all cultures were then subject to the cellular compartment fractionation procedure. cellular compartment fractionation cells were harvested by centrifugation at min/4600-g/4°c and separated into; medium fraction, soluble periplasmic fraction and cytosolic fraction by a modified version of that described by kipriyanov et al.. briefly, the medium fraction was obtained by an additional centrifugation of ml supernatant. to obtain the cytosolic and periplasmic fractions, the pellet was resuspended in μl ice-cold periplasmic extraction solution supplemented with μl lysozyme and μl rnase a before incubation for h at 4°c with rotation. the mixture was centrifuged, leaving the soluble periplasmic fraction as the supernatant. the pellet, containing the cytosolic fraction, was resuspended in μl ice-cold m tris-hcl, ph before addition of μl 4× sample buffer and subjected to rough pipetting to dissolve the pellet. for the large scale cultures, the same fractionation procedure was used after adjusting the reagent volumes × protein purification the periplasmic fractions isolated after 400-ml expression were diluted 1: in binding buffer and filtered through a μm sterile filter. the samples were purified by immobilized metal affinity chromatography on a his-traphp column as described in the protocol provided by the manufacturer. the eluted protein fractions were pooled, concentrated by amicon ultra- centrifugal filter units and dialyzed against storage buffer. protein concentration was determined by a nm on a nanodrop nd- apparatus based on calculated extinction coefficients. size exclusion chromatography was performed on an automated Δkta chromatography system, using μl of the imac purified protein to isolate monomeric sctcr. the concentration of protein in each fraction was determined as described above, followed by analysis on non-reducing and reducing sds-page and western blotting as described below. the monomeric fractions were pooled, concentrated by amicon ultra- centrifugal filter units followed by concentration determination as before. sds-page and western blotting identical volumes of fractionated samples isolated in parallel from a nm normalized e. coli cell cultures were separated by non-reducing sds-page on 4-12% bis-tris xt precast gels and blotted onto a polyvinylidene fluoride membrane in tris/glycine buffer at v for min using a semi-dry blotting apparatus. the membrane was blocked in pbstm before sctcr proteins were detected with anti his-hrp ab. the membrane was washed and developed with supersignal™ west pico substrate before exposed to biomax mr film. expression of rfkpa was detected after min stripping by restore™ western blot stripping buffer, and the membrane redeveloped as above using an anti-flag m2-hrp ab. samples of μg superdex purified sctcrs were analyzed in parallel with μg imac purified sctcrs by non-reducing and reducing sds-page followed by western blotting as described above. sctcr capture elisa the various mabs were absorbed to maxisorp microtiter plate wells at a concentration of μg/ml in × pbs, ph on at 4°c. the wells were blocked with pbstm for h at rt, and imac purified sctcr samples at a concentration of μg/well and superdex purified sctcr samples at a concentration of μg/well were added. all were allowed to react for h at rt before the captured sctcrs were detected with anti-his-hrp ab for h at rt. the wells were developed with μl of tmb soluble substrate, stopped by adding m hcl after min, and the absorbance read at a nm. circular dichroism spectroscopy cd spectra were recorded using a jasco j- spectropolarimeter. measurements were performed at 25°c using a quartz cuvette with a path length of cm. measurements were performed with a protein concentration of mg/ml for 4b2a sctcr vαβ and mg/ml for the control scfv ab fragment in 1× pbs, supplemented with mm nacl. samples were scanned times at nm/min, using a band width of nm. the response time was s, and the wave length range was 200- nm. the data were averaged and the spectrum of a sample-free control was subtracted. the secondary structure elements of the proteins were calculated from ellipticity data, using the spectral fitting methods, contin/ll, selcon and cdsstr, in the cdpro package. thermal denaturation was determined as the change in cd signal from heating. the temperature was controlled with a tpc-423s/l system. secondary structure prediction secondary structure element of the 2c tcr structure were predicted using the dssp and stride.  construct design the phog, pfkpen and pfkpei vectors drive sctcr expression from the lac promoter. in all cases, the sctcrs are fused to an n -terminal pelb signal sequence that directs protein translocation across the inner membrane of e. coli through the secb-dependent pathway to the oxidising environment of the periplasm. this oxidising environment is a prerequisite for generation of disulphide bridges within proteins such as sctcrs. the pfkpen and pfkpei vectors also carry the gene encoding the periplasmic chaperone fkpa. in pfkpen, recombinant fkpa expression is controlled by its native promoter, while pfkpei contains a dicistronic expression cassette where rfkpa gene transcription is controlled by the lac promoter. in all vectors, the sctcr cassette was constructed by genetically fusing vα and vβ encoding gene segments with a sequence encoding a synthetic linker. to gain insight into how domain orientation affects soluble expression, two versions with alternative domain orientation were made, such that either vα or vβ was expressed n-terminally. all sctcrs were fused to a c-myc- and a his-tag c-terminally, whilst rfkpa was fused to a c-terminal flag-tag. all constructs used in the current study are described in figure expression level and subcellular localization expression was performed in e. coli xl1-blue followed by cell fractionation and periplasmic expression analyzed by sds-page and western blotting, as described in the method section. as shown in figure 4b2a sctcr was not detected in the periplasmic fraction using anti-tag mabs in the absence of rfkpa over-expression, regardless of domain orientation and expression conditions. the 7a10b sctcr behaved like 4a10b. thus, both sctcrs failed to reach the periplasmic space, and rather aggregated in the cytosol. the growth characteristics of each culture were studied and the results are summarized in table the one expressing 4b2a vαβ sctcr showed sign of growth arrest. all others demonstrated overall robust growth and reached a final cell density similar to that of the xl1-blue control without plasmid. a a nm after on incubation of ml expression cultures b no growth of cultures after induction the chaperone fkpa has a highly beneficial effect on periplasmic expression and correct folding of toxic ab scfvs,, and we have recently reported a similar effect on phage display of sctcrs. thus, the effect of fkpa over-expression was assessed here. the clones that were picked for further study, all expressed high levels of fkpa as demonstrated by sds-page and western blot of whole cell lysates using anti-flag ab. the periplasmic levels of sctcrs in both configurations, vαβ and vβα, were detected under different expression conditions. the levels of rfkpa in the periplasm were also assessed. when expression was induced with iptg, a high yield of 4b2a sctcr in vβα orientation was observed, while sctcr with the opposite vαβ orientation was not. the highest yield of 4b2a vβα sctcr was obtained when rfkpa transcription was under lac promoter control. all four of these 4b2a sctcr producing cultures showed robust growth. the 7a10b sctcr was not detected or detected in negligible amounts in the periplasmic fraction regardless of orientation. the sctcr vαβ producing culture showed sign of growth arrest when rfkpa was transcribed from its endogenous promoter. it has earlier been reported that a reduction of the iptg concentration has a positive effect on periplasmic yield of soluble protein expressed from a lac promoter. indeed, when expression was performed without iptg, the periplasmic yield of 4b2a vαβ increased considerably. the yield of 4b2a vβα, on the other hand, decreased. the yield of 7a10b sctcr in both domain orientations increased. all cultures reached high and similar final cell density. analysing periplasmic fkpa levels, this was equal to or higher than that observed in the presence of iptg in all cases, except when expressed from the lac promoter in the vector encoding 4b2a sctcr vβα. this was in line with the fact that very little 4b2a sctcr vβα was found in the periplasmic space in the absence of induction. addition of m sucrose to the growth medium has been shown to increase the yield of secreted proteins, either in the periplasm or culture medium. to investigate if the soluble sctcr yield in the periplasmic fraction could be further increased, the effect of sucrose addition was assessed both in uninduced and iptg induced cultures. neither the 4b2a nor 7a10b sctcr levels increased after sucrose addition. the expression patterns were identical to that observed without sucrose addition, and no increased leakage to the culture medium was observed. taken together, overall good yields of periplasmic 4b2a sctcr were seen in the presence of periplasmic rfkpa expression. this was observed from both pfkpen and pfkpei plasmids; for vαβ, when expression was performed in the absence of iptg and for vβα, when expression was performed in the presence of iptg. for 7a10b sctcr, the overall highest yield was seen from the pfkpen plasmid in the absence of iptg for both the vαβ and the vβα configuration. protein purification the four sctcr variants, 4b2a and 7a10b in both domain orientations, were expressed at large scale under conditions shown to give good small scale periplasmic yields. protein fractionation was performed as described above, followed by imac purification. eluted fractions containing protein were pooled and concentrated. the production yields after concentration is given in table and were and mg/l for the 4b2a vαβ and vβα variants, respectively. very low yield of the 7a10b variants were obtained. thus, the two 4b2a samples only were further analyzed by size exclusion chromatography, followed by non-reducing and reducing sds-page of eluted fractions. importantly, the vαβ format had more than double the amount of monomeric sctcr compared to the vβα format. however, both formats were produced as polymers that appeared to be composed of both non-covalent and covalent aggregates. taken together, the overall best yield of monomeric sctcr was from the 4b2a clone in the vαβ orientation. this format yielded mg per l culture of active purified monomeric material, which is comparable with or better than amounts acquired by others using periplasmic expression systems. importantly, the hucκ fusion format reported by maynard et al requires downstream proteolytically removal of the hucκ moiety, hence the actual soluble sctcr yield will be equal to or lower than with our approach. moreover, the absence of this hucκ fusion resulted in wastely reduced sctcr yield. a estimated periplasmic yield of total soluble sctcrs after imac purification. b estimated periplasmic yield of monomeric soluble sctcr after size exclusion chromatography. c values implemented from maynard et al, sctcr-human c kappa fusion format d values implemented from maynard et al, sctcr format nd; not determined folding and secondary structure analysis elisa the conformational integrity of the imac and size exclusion chromatography purified 4b2a sctcrs was analyzed using two v-gene specific mabs in a capture elisa. furthermore, 7a10b sctcrs were produced in several parallel small scale cultures, that were pooled, imac purified and analyzed in capture elisa. the results show that differences exist in mab reactivity between the sctcrs with regard to domain orientation. for both 4b2a and 7a10b sctcr, the vαβ sctcrs exhibited stronger mab reacitivity than the vβα counterparts. both anti-vβ mab f and clonotype-specific gb bound the purified 4b2a sctcrs fractions. gb bound the two molecules equally well, whilst f bound better to vαβ than to vβα, likewise, the imac purified 7a10b vαβ sctcr was detected by the vβ6-specific 44-2- and rr4- mabs, whereas no binding was observed in the vβα domain orientation. structural analysis by cd spectroscopy sctcr vαβ 4b2a and a control scfv ab fragment were subjected to cd spectroscopy at 20°c and 56°c as shown in figure and the spectra obtained analyzed using contin/ll, selcon and cdsstr. the obtained structure data from the three fitting techniques were similar. however, contin/ll gave slightly lower rmsd ratios than cdsstr. the results from contin/ll are thus given in table the cd data analysis indicated a 47% and 42% β-sheet content as well as a 5% and 5% α-helix content in the scfv control and 4b2a sctcr vαβ molecules, respectively. the spectra of both 4b2a sctcr vαβ and the control scfv ab fragment are hence typical of a protein with high degree of β-sheet structure characteristic for the ig-fold. this is consistent with the secondary structure element prediction given by dssp and stride for the 2c tcr structure, which calculates a 49% β-sheet content and a α-helix content of %. the degree of secondary structure content decreased as the temperature was raised towards 56°c as seen in table and figure 6c, indicating thermally induced unfolding of the protein. a the secondary structure elements were estimated by contin/ll from the cd spectrums presented in figure the experiment was repeated twice, and representative results are shown. discussion the aim of the current study was to generate a general expression system for soluble sctcrs. the 4b2a and 7a10b sctcrs used have previously been successfully expressed as fusions to piii on filamentous phage. based on that study, the current report focuses on the soluble sctcr format and investigation of protein yield and sub-cellular localization using a bacterial system compatible with the e. coli host used in sctcr phage display, namely xl1-blue. chaperones are known to enhance expression yields as they facilitate folding, prevent aggregation, reactivate aggregates and reduce protein degradation. bothmann and plückthun identified two periplasmic factors, namely fkpa and skp, which increased the functional periplasmic yields of toxic ab scfvs, while already well-expressing clones were unaffected. fkpa is a periplasmic peptidyl-propyl cis,trans-isomerase, but the effect was also striking for scfvs that did not contain cis-prolines. the positive effect rather seemed to be a result of the ability of the chaperone to prevent premature aggregation of early folding intermediates as well as reactivation of inactive proteins. skp belongs to a family of cavity-containing chaperons and works by protecting its substrate from aggregation within this cavity. skp was recently reported to have an effect on sctcr expression. however, co-expression of skp and fkpa showed no synergistic effect on scfv yield. rather, co-expression had the same effect as fkpa alone. we have previously reported that over-expression of rfkpa normalized the display level of sctcrs-piii fusions in phage display, while skp had only minor effects. in the present study, we therefore focused on fkpa co-expression. without chaperone co-expression, the sctcrs were trapped in the cytosol despite the fact that all constructs were equipped with a pelb leader sequence directing transport to the periplasm. when fkpa was co-expressed, we observed a positive effect on growth rate. moreover, a profound effect on the accumulation of soluble 4b2a and 7a10b sctcrs in the periplasm was also seen. this was the case whether rfkpa was expressed from its native promoter or the lac promoter. these initial results hence showed that fkpa has an ability to significantly increase the periplasmic expression yield of the tcrs. the effect of domain orientation on functional sctcr fragments has been reported earlier, and show the same trend as we see here, namely that the vα-linker-vβ domain orientation increases the functional fraction of sctcr fragments. generally, the most unstable fragment should be put n-terminally and the most stable fragment c-terminally. this prevents rapid folding of the n-terminal domain followed by abortive periplasmic translocation caused by a partly unfolded c-terminal domain. the trbv13- segment used in the 4b2a sctcr, is considered a particularly stable fragment. the results in this report hence fit well with the results obtained by others. culture conditions, such as induction and growth in the presence of certain sugars, have been shown to influence the production yield and sub-cellular localization of proteins. we therefore investigated the effect of sucrose addition to the medium as well as the effect of adding or removing iptg. the results showed that 4b2a tcr in the vβα orientation was expressed well under standard conditions, that is, in the presence of iptg, but without addition of sugars. in contrast, 4b2a tcr vαβ and 7a10b in both domain orientations were expressed poorly. removal of iptg from the growth medium restored the expression of these three molecules. without iptg residual transcription still occurs through the leaky lac promoter, but with decreased translation and hence folding load. this was found to be beneficial for the sctcrs. it has previously been reported that growing cells in the presence of certain sugars, raffinose or sucrose, can reduce the aggregation of recombinant proteins in the e. coliperiplasm. these sugars are small enough to diffuse through the inner membrane to the periplasm, causing an increase in the osmotic pressure resulting in enlargement of the periplasmic space, and hence a decrease in the local protein concentration. however, the addition of sucrose had no effect on the yield of sctcr in the periplasm. possibly, the over-expression of rfkpa prevents premature aggregation so efficiently that the addition of sucrose has little or no detectable effect. when large scale expression using optimalized expression conditions for each of the sctcrs was performed mg/l amounts were obtained from both 4b2a sctcrs after purification. the 7a10b derived sctcrs showed an altered expression profil after up-scaling, which has also been reported by others for antibody scfv fragments. however, by pooling the periplasmic fraction from several small scale cultures, yields of μg/l were obtained. thus, the 4b2a derived molecules only were chosen for studies of how domain orientation influences the yield of the monomeric fraction as well as folding. we found that the sctcr with the vαβ domain orientation had a larger monomeric peek than the one with the vβα domain orientation. aggregates appeared to be formed after misfolding due to erroneous disulphide bond formation as well as through surface exposed hydrophobic patches. the affinity of the monomers for their cognate pmhc ligand, is too low for detection by flow cytometry. we therefore used binding to several well characterized mabs as an indirect measurement of structural integrity and functionality. the same mabs have been used earlier to characterize the same sctcrs displayed on phage or expressed as tcr-ig fusions. in both cases, binding to mabs correlated with the ability of the tcr moiety to bind i-ed expressing murine a lymphoma cells that had been loaded with the cognate λ peptide, and binding required a multivalent format. importantly, the current report shows differences in mab reactivity with regard to domain orientation, and the vαβ format was superior to the vβα format. furthermore, the secondary structure data given by cd measurements shows that the sctcr vαβ has a native-like fold that in conjunction with the binding activity of the mabs strongly indicates functionality.  the work demonstrated that the presence of the chaperone fkpa, expression conditions as well as domain orientation greatly influence the yield and sub-cellular localization of soluble tcrs. over-expression of fkpa in the periplasm is a prerequisite for soluble periplasmic localization of the sctcr, and absence of inducer further improved the periplasmic yield of formats which were poorly expressed under standard conditions. addition of sucrose to the medium did not influence the yield. comparing sctcr molecular design, the format with vαβ domain orientation gives a better expression profile with regard to monomeric protein yield than the vβα format, and the binding to specific mabs is stronger. furthermore, the purified 4b2a sctcr vαβ exhibited well resolved secondary structure elements typical for the ig fold topology indicating a correctly folded tcr moiety. the final protein yields obtained using our optimized approach perform equal or better than previously reported periplasmic expression systems and this was achieved without fusions or aa modifications of the tcr moiety. we anticipate that this system may enable rapid production of a wide range of soluble tcrs, and thereby simplify the screening of a new generation of recombinant tcrs generated by protein engineering and display techniques such as phage-display. authors' contributions ksg did most of the experimental work, participated in the design of the study and drafted the manuscript. el gave initial technical guidance. pek gave guidance on the cd analysis. bb provided key reagents, participated in the design of the study and in drafting the manuscript. gÅl designed and constructed the expression vectors, supervised the study and participated in drafting and finalizing the manuscript. is supervised the study, provided funding and finalized the manuscript. all authors approved the final manuscript. supplementary material additional file 1 primer overview. primers used to isolate the fkpa gene orf. click here for file acknowledgements the authors would like to thank sathiyaruby manicam vladivelu for excellent technical assistance. this work was supported by the norwegian research council.
periplasmic expression enables disulphide bond formation, which is a prerequisite for the sctcr to reach its correct fold. expression without iptg induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. the general expression regime presented here allows for rapid production of soluble sctcrs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering. the vαβ domain orientation was far superior to the vβα domain orientation regarding monomeric yield of functionally folded molecules. the effect of 1) over-expression of the periplasmic chaperon fkpa, 2) culture conditions and 3) molecular design was investigated. elevated levels of fkpa allowed periplasmic soluble sctcr expression, presumably by preventing premature aggregation and inclusion body formation.
 efficient expression systems exist for antibody molecules, which allow for characterization of large numbers of individual ab variants. in contrast, such expression systems have been lacking for soluble t cell receptors. attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. here we present an optimized periplasmic bacterial expression system for soluble single chain tcrs.  the effect of 1) over-expression of the periplasmic chaperon fkpa, 2) culture conditions and 3) molecular design was investigated. elevated levels of fkpa allowed periplasmic soluble sctcr expression, presumably by preventing premature aggregation and inclusion body formation. periplasmic expression enables disulphide bond formation, which is a prerequisite for the sctcr to reach its correct fold. it also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. expression without iptg induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. shaker flask yield of mg levels of active purified material was obtained. the vαβ domain orientation was far superior to the vβα domain orientation regarding monomeric yield of functionally folded molecules.  the general expression regime presented here allows for rapid production of soluble sctcrs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering. 