| Breast cancer is a major cause of morbidity and mortality in women in theUnited States , China and many other countries. Several candidate biomarkermolecules correlated with breast tumors have been suggested and studied, suchas KLK12, AZU-1, P7 antigen and TSP50 (testes-specific protease 50) .The TSP50 gene was isolated from a human testes cDNA library by using agenomic DNA probe, BR50. BR50 was isolated by a modified representationaldifference analysis technique due to its hypomethylated feature in a breastcancer biopsy. Northern blot analysis of RNA from multiple normal humantissues indicated that this gene was highly and specifically expressed in thetestes, and it is a tissue-specific gene. TSP50 gene product shares a similarenzymatic structure with many serine proteases. Amino acid alignment withseveral serine proteases revealed that although TSP50 shared two criticalcatalytic triads, histidine and aspartic acid, the most critical catalytic site, serine,was substituted with threonine, which is different from traditional serineproteases;it could therefore be classified as a novel protease [6]. However, inaddition to expression in normal testes, the TSP50 gene was abnormallyactivated and expressed in a vast majority of breast cancer patients tested [6].Earlier studies have suggested that the normal function of the TSP50 geneproduct might be involved in the human reproductive pathway, but that its upregulation could be linked to tumorigenesis in human breast cancer. Thesurprisingly high positive detection rate of the TSP50 protein in breast cancercould make it an attractive molecular marker for this disease [6].Based on the information above, we wished to acquire the genefragments of scFvs against TSP50 using phage display antibody librarytechnology, and clone them into the expression vector pPELB and expressthem in E.coli Rosetta. If the expressed proteins show a specific reaction withTSP50 molecules by immunological examination, it will be very beneficial tothe investigation of the function of TSP50 in the development of breastcancer and its relevance to the treatment of breast cancer.This paper is the first account of the selection of recombinant antibodyfragments for TSP50 from a naive phage display library, i.e. one constructedwithout prior exposure to antigen. The library was prepared without theinvolvement of laboratory animals, using a framework based on a pairing ofVH and VL chains most common in the human immune response. If the scFvantibodies can be used as inhibitors or target carriers to treat breast cancer,the human antibody response against mouse protein can be avoided,enhancing therapeutic utility.Materials and methodsSupplies. Reagents and their suppliers are as follows: mouse anti-M13phage, anti-hexa-histidine monoclonal antibody and Fast Flow Sepharose(Pharmacia Biotech, Sweden);Tryptone, Yeast Extract Gibco, BRL);Immunotubes (Maxisorp, Nunc);CM Fast Flow,Chelating Sepharose FastFlow and Sephadex G-25 (Pharmacia, Sweden);tetra methyl benzidine(TMB), SDS, PMSF, immidazole, IPTG (Isopropyl-β -D-thiogalactopyranoside), N,N-methylene-diacrylamide and acrylamide (Sigma,USA);96-well flat-bottomed ELISA plates (Becton Dickinson. USA);horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbitIgG (Santa Cruz, USA);restriction enzymes, NcoI and NotI (TaKaRa, China);protein molecular weight markers (Bio Rad, USA and Shanghai BiochemicalInstitute, China);ELISA reader (ELX800, BIO-TEK Co. USA). Allchemicals were of the highest purity available or otherwise of a quality notedin the text.Phage antibody library, vector and bacterial strains. Human syntheticVH+VL scFv library, helper phage M13K07 and E. coli TG1 used in this studywere generous gifts from Fiona Sait, Centre for Protein Engineering, MedicalResearch Council (MRC), University of Cambridge, England. This scFvphagemid library contains synthetic V-gene (VH-VL) from lox library vectors[7] recloned into the pHEN2 phagemid vector and the library size is 1.47×108 phagemid clones in E. coli TG1 cells and has a high proportion of functionalantibody with 96% of clones containing inserts (These are also commerciallyavailable from MRC and Pharmacia Biotech;see provided website).Vector pPELB and E. coli Rosetta were generous gifts from Dr. SuiDexin, University of Michigan.Antigen used for screening. The antigen, TSP50-specific peptide,pep-50, was synthesized at the Facility of Molecular Genetics, NorthShore-Long Island Jewish Research Institute. The sequence of pep-50 wasIle-Trp-Arg-Asp-Val-Ile-Tyr-Ser-Val-Arg-Val-Gly–Ser-Pro-Trp-Ile-Asp-Gln-Met-Thr-Gln-Thr-Ala-Ser-Asp-Val-Pro-Val-Leu-Gln-Val-Ile-Met-His-Ser-Arg-Tyr-Arg-Ala-Gln-Arg-Phe-Trp-Ser-Phe-Val-Gly-Gln-Ala-Asn,corresponding to the 156–206 aa position, which was a nonhomologoushydrophilic region within the catalytic domain. Pep-50 is a critical catalyticsite of TSP50 different from other traditional serine proteases and readilybinds to antibodies when used as a target antigen. The activity of TSP50 canbe inhibited when the prepared scFvs bind to pep-50, allowing investigationof the role of TSP50 in tumorigenesis in these studies. Jidong Shan et al. haveprepared anti-TSP50 polyclonal antibody AT-50 using synthesized pep-50 asan antigen and applied it in their studies [6]. Up to now, full length TSP50 isnot available;therefore,we used pep-50 as a target antigen substituting forthe full length TSP50 to screen scFvs from the antibody library.The rescue of phage from the cells The whole of the bacteriallibrary stock (about 1×10 10 clones, stored at -80℃ ) in 500 ml of 2×TYculture medium containing 1% glucose and 100 ug/ml ampicillin wasincubated with shaking at 37℃ to an OD600 of 0.4. M13K07 helper phage (1×1012 pfu ) were added to 50 ml of the culture and the mixture was incubatedwithout shaking for 30 min at 37℃ [8]. Infected cells were pelleted and thenresuspended in 100 ml 2×TY broth containing 1% glucose and 100 ug/ml23ampicillin and 50 ug/ml kanamycin, and incubated overnight with shaking at30℃. PEG/NaCl (20% polyethylene glycol 6000, 2.5 M NaCl) in a 1/5volume was used to precipitate phage particles from the culture supernatantas described previously [9]. The pellet was resuspended in 2 ml PBS andconcentrated at 11, 600 g for 10 min in a microcentrifuge to remove most ofthe remaining bacterial debris. The phage supernatant was stored at 4℃ forshort term storage or in PBS, 15% glycerol for longer term storage at -70℃.To determine the titer of the phage stock, 1ul phage was diluted in 1 ml PBSand 1ul of this dilution was used to infect 1 ml of TG1 at an OD600 of 0.4-0.6and incubated for 30 min at 37℃. Fifty ul of this, 50 ul of a 1:102 dilution and50 ul of a 1:104 dilution were plated on TYE plates containing 100 ug/mlampicillin and 1% glucose and grown overnight at 37℃. The titer of thephage stock was 5.6 ×1012 pfu / ml determined according the number ofTG1 clones on the TYE plates.Affinity selection of anti-TSP50 phage antibodies A 30×10 mmImmunotube (Maxisorp, Nunc) was coated with 1 ml pep-50 antigen, at 10ug/ml in phosphate-buffered saline (PBS, pH 8.5), and incubated overnight at4℃, washed 3 times with PBS and blocked with 3% BSA-PBS at 37℃ for 2h. The phage stock (5.6 ×1012 pfu) was added to 1 ml 3% BSA-PBS in thetube, and incubated for 30 min at RT, rotating continuously on anunder-and-over turntable, and then a further 60 min at 37℃ without rotation.The tube was washed 20 times with PBS containing 0.1% Tween 20, and24bound phage eluted for 10 min using 1ml 100 mM triethylamine as describedabove [10]. During the elution, tubes were previously prepared with 0.6 ml 1M Tris-HCl, pH 7.4, to add the eluted 1 ml phage for quick neutralization.Half of the eluted phage (0.8ml) was added into 9.2 ml exponential phaseTG1 cell culture suspension in 2×TY broth, and any remaining bound phagein the tube eluted by adding 100 ul 1M Tris-HCl, pH 7.4, and 4 ml TG1culture suspension. Both TG1 cultures were incubated for 30 min at 37℃without shaking to allow for infection. The cells were pooled, concentratedby centrifugation and plated on TYE agar [11] containing 1% glucose and 100ug/ml ampicillin. After incubation overnight at 30℃, colonies were scrapedinto 5 ml of 2×TY broth containing 1% glucose, 100 ug/ml ampicillin and20% glycerol, and stored at -80℃ , Glycerol stock (100ul) was theninoculated into 50 ml of 2×TY broth containing 1% glucose and 100 ug/mlampicillin, and incubated with shaking at 37℃ to an OD600 of 0.4. Selectedphages were then rescued from the culture as described above. Selection wasrepeated another 3 rounds with the following modifications: a) pan 2, theImmunotube blocked with 3 % glycine-PBS;and b) pans 3-4 used pep-50 at500 and 25 ng/ml, respectively i.e., the concentration of pep-50 used forcoating was reduced from 10ug/ml (pan 2) to 25 ng/ml (pan 4).Screening and selection of phage antibodies. Individual coloniesfrom pan 4 were grown in 96-well plates and phage antibodies were rescuedwith M13K07 following the description above. The specificity of phagesupernatant for binding to pep-50 was then determined using indirect ELISA.Indirect ELISA was performed to assess the binding ofpolyethylene glycol-precipitated phage at each round of panning, and phageclones binding to pep-50, but not to either BSA or glycine alone, were pickedThe wells of a 96-well flat-bottom ELISA plate were coated with 50ul ofpep-50 (5ug/ml) overnight at 4℃, and then washed with PBS containing0.1% Tween 20. The plates were blocked with 200 ul per well of 3%BSA-PBS at 37℃ for 2h and then washed as described above. Phageprecipitated at each round of panning (50 ul), used as the first antibody, wereadded to the wells in 3%BSA-PBS. The plates were then incubated at 37℃for 2h, and washed three times with PBS containing 0.1% Tween 20 prior toadding 50ul per well of a 1:1000 dilution of anti-M13 monoclonal antibodyas the second antibody and incubation at 37℃ for 1h. Bound antibodies weredetected with an HRP-Goat anti-mouse IgG and tetramethylbenzidine(TMB)-H2O2 substrate. The reaction was terminated with 50 ul per well of 1M H2SO4, and the optical density was read at 450nm [11].Construction of the recombinant plasmids. The antibody's VH and VLgenes from selected clones were amplified using polymerase chain reaction(PCR), and primers corresponding both to the 5' and 3' sequences of the vectorpHEN2(5'-CAG GAA ACA GCT ATG AC -3'and 5'-GAA TTT TCT GTATGA GG -3' from TaKaRa Co.) and the phagemid DNA prepared from theselected clone as template. The products were sequenced by TaKaRa Co. inboth directions using the same primers (5'-CAG GAA ACA GCT ATG AC -3'and 5'-GAA TTT TCT GTA TGA GG -3' from TaKaRa Co.) [12, 13]. PhagemidDNAs from the clones found to have different complementary determiningregions by sequencing were digested with Nco I and Not I, and then the scFvgenes were cloned into the similarly digested soluble expression vector pPELB(a gift of Dr. Sui Dexin, Univ. of Michigan).Expression of scFvs in E.coli Rosetta. The recombinant plasmids weretransformed into E. coli Rosetta by electroporation. Transformed E. coliRosetta clones were incubated in 500 ml 2×YT containing 100 ug/mlampicillin, and 34 ug/ml chloramphenicol at 37℃. 500 ul 1M isopropylthiogalactopyranoside (final concentration 1 mM IPTG) was added into theculture when the OD600 reached 0.6. Shaking was continued at 30℃ for afurther 4 h [14]. Cells were harvested by centrifugation at 6000 rpm at 4℃ for10 min, and either stored frozen at -70℃ or disrupted by sonication asdescribed below and used directly for SDS-PAGE analysis as describedpreviously [14].Purification of the bacterial scFvs. The scFv proteins acquired abovewere purified as follows [14, 15]. Cells were resuspended in 50 mM PBS, pH6.5, 1 mM PMSF, to a final ratio of approximately 5 ml lysis buffer per gramof cells. Cell suspensions were sonicated with middle pulse extension on iceand supernatants were obtained by centrifugation at 12000×g at 4℃ for 15min and filtered using a 0.45 um filter. Ion-exchange chromatography wascarried out on the column packaged with CM Fast Flow resin. The columnwas equilibrated with the buffer A (50 mM PBS, pH 6.5). Sample was loadedonto the column overnight at 4℃ and eluted by a step NaCl gradient asfollows. Weakly bound proteins were removed by adding 400 mM NaCl inbuffer A. ScFvs were eluted by raising the concentration of NaCl to 1 M. Theeluent containing scFvs was purified further with immobilized metal ionchelate affinity chromatography (IMAC) via the hexa-histidine tail on 1 mlHiTrap chelating column using Ni2+ charged (Amersham-Pharmacia) [15]. Thecolumn was equilibrated with buffer B (50 mM PBS, pH 7.5, 300 mM NaCl).Sample (5-10mg protein/ml gel) was loaded onto the column and eluted asfollows. Nonspecifically and weakly bound proteins were removed by addingimidazole 80 mM in buffer B. ScFvs were eluted by raising the concentrationof imidazole to 100 mM. The flow rate was 1 ml/min. After elution, EDTAwas added to fractions at a final concentration of 1 mM in order to preventthe protein from precipitating upon removal of the imidazole by dialysis. It ispossible that the addition of EDTA scavenges leached Ni2+ from the Hitrapresin and prevents the His-tagged protein from divalent cation mediatedcross-linking and precipitation. The concentration of the purified scFvproteins was determined with Lowry's method [16]. Purified antibodyfragments were dialysed against PBS and stored in 15% glycerol at -20℃prior to use.Electrophoresis and western blot. SDS-PAGE in 15% gels using aTris-glycine buffer was used to monitor the purification during thechromatographic procedures. ScFvs were confirmed by 15% SDS-PAGE andwestern blot according to routine methods [17, 18]. Briefly,the proteinscontained in the supernatant and in the chromatographic fractions wereseparated by electrophoresis as above and transferred onto a nitrocellulosemembrane. The membrane was blocked with TTBS (100 mM Tris-HCl, 150mM NaCl, 0.2% Tween 20 , pH 7.5) containing 3% BSA overnight at 4℃.This membrane was incubated for 1 h at 37oC in TTBS and washed at roomtemperature by TTBS. The membrane was then incubated in 10 ml of TTBScontaining 50 ug of monoclonal antibody against hexa-histidine for 1 h at37oC, and washed in TTBS four times. The membrane was then incubatedwith horseradish peroxidase (HRP)-labeled goat anti-mouse IgG for 1 h at37oC. After four washes with TTBS for 15 min, the bound antibodies wererevealed using the tetrammine benzidine (TMB) solution containing 0.03%H2O2.Sandwich ELISA. The wells of a 96-well ELISA plate were coated,respectively, with scFv-A1 and scFv-A11 at a concentration of 10 ug/ml in PBSovernight at 4℃. The plates were blocked with 200 ul per well of 3% BSA inPBS at 37℃ for 2h. Pep-50 diluted in PBS-BSA to concentrations rangingfrom 200 to 0.001 ug/ml and 3% BSA in PBS as negative control was,respectively, added to the wells and incubated at 37℃ for one h. The wellswere washed three times with PBS containing 0.1% Tween 20, and then rabbitanti-pep-50 polyclonal antibody was added and incubated at 37℃ for one hour.Bound pep-50 was detected with an HRP-Goat anti-rabbit IgG and TMB-H2O2solution. The reaction was terminated with 50 ul per well of 1 M H2SO4, andthe optical density was read at 450 nm.ResultsSelection of single-chain antibody against TSP50ELISA of phage rescued from the original library (pan 1) and pans 2-429indicated an enrichment of binders for pep-50 during panning, but not for eitherBSA or glycine alone. The enrichment of phage antibodies to BSA wasminimized by alternating BSA used to block Immunotube into glycine betweenpans 1 and 2. The stringency of selection for higher affinity pep-50 binders wasincreased further by decreasing the pep-50 concentration 20-fold between pans2, 3 and 4 [10]. One hundred phage clones were randomly picked from the fourthround of panning and tested for specific binding to pep-50 by indirect ELISA.Three clones (A1, A11 and C8) showed strong binding only to pep-50;theiraffinities to pep-50 are 1.7×102, 1.3×10 and 1.1 L/mol, respectively. Theresults of DNA sequencing showed that they have 723-727 bp, consisting of330-333 bp of light chain variable region gene, 341-344 bp of heavy chainvariable region genes, and 45 bp of linker genes. However, they have differentcomplementary determining regions. Two unique clones (A1 and A11) wereselected according to their affinities to pep-50 and the results of sequencing andtheir molecular weights were about 31-32 kDa (predicted from their amino acidcomposition).Clone and expression of scFvs A1 and A11 in E.coli RosettaThe phagemid DNAs from the clones found to have differentcomplementary determining regions by sequencing were digested withNotI/NcoI and the scFv genes were cloned into expression vector pPELB,containing many polyclonal sites and leading sequence of making solubleexpression, digested with the same two enzymes. The constructed expressionvectors pPELB-A1 and pPELB-A11 were transformed into E.coli Rosetta.Before expression of induction, the recombinant plasmids in transformedbacterial cells were examined by digesting the plasmids extracted from thepositive clones with the two enzymes of NotI and NcoI, and sequencing. Theresult of DNA electrophoresis demonstrated that the target gene fragment of750 bp was released by digestion (Fig 1), and the results of DNA sequencingof the plasmids in the positive clones indicated that the coding sequences ofVH, VL, linker and leader were consistent with those before the recombination.Figure 2 demonstrates that scFv fragments were expressed as soluble proteinin E.coli Rosetta.Characterization of the pep-50 binding scFvsThe scFvs were purified by CM-Sepharose Fast Flow and IMAC. Theirpurity was tested by SDS-PAGE, followed by immunoblot experiments.Figure 3 shows electrophoretic analysis of the fractions resulting from thepurification. The final products showed a single band and their molecularweights were 31 and 31.4 kDa compared with the molecular weight ofmarkers. The molecular mass accorded with predicted molecular weightaccording to the sequencing results. The yields of soluble scFv-A1 andscFv-A11 after purification were about 4.0 and 5.0 mg of protein from 1 L ofculture, respectively. Figure 4 shows the results of western blotting of scFvspurified by CM-Sepharose Fast Flow and IMAC and the negative controlRosetta transformed with pPELB. The antibody used in this analysis detectsthe C-terminal His-tag fused to scFv gene.Binding of soluble scFvs to pep-50The binding of the expressed single-chain antibody fragments to freepep-50 was determined by sandwich ELISA using scFv-A1 and scFv-A11 asthe capture antibody, and rabbit anti-pep-50 polyclonal antibody as thedetecting antibody. The two antibody clones bound to free pep-50 with thelower detection limit of 4 uM scFv-A11 and 20 uM scFv-A1. In theory,theScFvs can react with the full length TSP50, but as we have not acquired thefull length TSP50 to date, it should be explored in later studies.DiscussionsIn this study, phage display antibody library technology was applied togenerate anti-TSP50 antibody fragments. The main advantage of this techniqueis that human antibodies can be produced much easier than human hybridomas.These can be then developed for therapeutic use without the need to humanizemouse Mabs. Furthermore, by changing the selection conditions, the antigenbinding properties of antibody phages can be influenced, e.g., by selection forhigh-affinity antibodies or specificity for a predetermined epitope [19, 20]. Therandom pairing of V domains has the potential to generate artificial antibodyspecificities and could overcome the problem of the immune dominance ofsome antigens and epitopes in mice that limit the spectrum of specificitiesobtained with hybridomas [21]. Furthermore, using this technique, the procedurewas faster, easier and considerably less expensive than the generation ofpolyclonal and monoclonal antibodies against the antigen. The antibodyacquired in this study is single chain variable fragment (scFv), which is thesmallest antibody fragment containing a complete antigen binding site. Incomparison to the much larger Fab', F(ab')2 and IgG forms of monoclonalantibodies, scFv have lower retention times in non-target tissues. Unlikeglycosylated whole antibodies, scFv antibodies can be easily produced inbacterial cells as functional antigen binding molecules.A humanized monoclonal antibody against HER-2 e.g., Herceptin andscFv antibodies with biological function have been developed for studies on therole of growth factor receptor pathways in breast cancer and leukemia. Some ofthem have been used as therapeutic ally [22]. They bind to the extra cellularportion of transmembrane receptors which is over expressed in subsets oftumors, and inhibit cell proliferation in a dose dependent manner [22, 23]. Thefunction of TSP50 in the development of breast cancer is still unknown. Thus,the scFvs selected in this study could be valuable reagents for immunoassay ofTSP50 as well as the investigation of the function of TSP50 in the developmentof breast cancer. They were also eventually used as therapeutic agents for breastcancer.In conclusion, we have panned the scFv antibodies against TSP50 from anaive phage display antibody library using pep-50 as target antigen andsuccessfully expressed the genes of scFvs in E.coli Rosetta. These antibodieshave the ability to specifically recognize pep-50 with high affinity. If a genemodification to the scFvs can be accomplished using error-prone PCR,mutation strains, chain shuffling or DNA shuffling [11, 24], the antigen-scFvaffinity can be further increased. Our results indicate that synthetic humanantibody libraries can be useful tools for the selection of antibodies against anonhomologous hydrophilic region within the catalytic domain of theprotease. We are currently investigating the function of TSP50 in thedevelopment of breast cancer using selected scFvs, and the clinicalapplication of the scFv antibodies in the treatment of breast cancer.AcknowledgementsWe thank Fiona Sait from M.R.C., Cambridge, UK, for providing thephage library. We thank Dr. Sui Dexin from University of Michigan forproviding Vector pPELB and E. coli Rosetta.Fig 1Figure 1. Agarose (1.2% ) gel electrophoresis of the digested A1-pPELBand A11-pPELB with NotI/NocI. Lane 1, the target fragment released fromthe A1-pPELB digested with NotI/NocI;lane 2, the target fragment releasedfrom the A11-pPELB digested with NotI/NocI.Fig 2Figure 2. The expression of soluble scFvs under the preferred cultureconditions. Lanes 1, 2, total soluble protein in the lysate of Rosettatransformed by pPELB-A1 and induced with IPTG in lane 2 and withoutIPTG induction in lane 1;lane M, protein molecular weight markers;lanes3, 4, total soluble protein in the lysate of Rosetta transformed bypPELB-A11 and induced with IPTG in lane 3 and without IPTG inductionin lane 4;lane 5, total soluble protein in the lysate of Rosetta transformedby pPELB and induced with IPTG.Fig 3Figure 3. 15% SDS-PAGE analysis of the fractions eluted from CM andIMAC columns. Lanes 1, 4, 25 ug of protein containing scFv-A1 andscFv-A11 eluted from CM-Sepharose Fast Flow with 20 mM PBS 1.0 mMNaCl, pH 6.5;lanes 2, 3, 8 ug of scFv-A1 and scFv-A11 eluted from IMACwith 100 mM imidazole;lane M, low molecular weight markersFig 4Figure 4. Western blot analysis of scFv proteins purified byCM-Sepharose Fast Flow and IMAC. The scFv proteins were purified,analyzed by 15% SDS-PAGE, transferred to nitrocellulose, and then detectedby monoclonal antibody against hexa-histidine. The lysate of Rosettatransformed by pPELB was used as a negative control (lane5). Lanes 1, 4, 25ug of proteins containing scFv-A1 and scFv-A11 eluted from CM-SepharoseFast Flow with 20 mM PBS 1.0 M NaCl, pH 6.5;lanes 2, 3, 8 ug of scFv-A1and scFv-A11 eluted from IMAC with 100 mM imidazole. |
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