The Korean Journal of Microbiology (2011) Vol. 47, No. 4, pp. 328-334 Copyright c 2011, The Microbiological Society of Korea Leucine Zipper Motif 를이용한닭의재조합이량체 Single-chain Fv (ScFv) 항체의개발 박동운 1 김언동 1 김성헌 1 한재용 2 김진규 1 * 1 창원대학교자연과학대학미생물학과, 2 서울대학교농업생명과학대학농생명공학부 WCU 바이오모듈레이션전공 The Development of Dimerized Chicken Recombinant Single-chain Fv (ScFv) Antibody Using Leucine Zipper Motif Dong Woon Park 1, Eon Dong Kim 1, Sung Heon Kim 1, Jae Yong Han 2, and Jin-Kyoo Kim 1 * 1 Department of Microbiology, College of Natural Sciences, Changwon National University, Changwon 641-773, Republic of Korea 2 WCU Biomodulation Major, Department of Agricultural Biotechnology, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea (Received September 27, 2011 / Accepted November 25, 2011) Leucine zipper motif consists of multiple periodic leucine residues, which forms amphipathic alpha helix. The hydrophobic nature of leucine zipper motif can dimerize proteins which contain this motif. Leucine zipper motif addition at C-terminus of single-chain Fv (ScFv) antibody induces its dimerization. Since the dimeric ScFv antibody contains two antigen binding sites (bivalency) like Y-shaped complete antibody, it could increase avidity. As a result, it could show higher antigen binding activity than monomeric ScFv antibodies. Based on this concept, monomeric chicken 8C3 ScFv antibody previously developed from chicken hybridoma was dimerized by the addition of leucine zipper motif at C-terminus of ScFv antibody. The dimeric 8C3 ScFv antibody specifically reacted with Eimerian sporozoite which causes Avian Coccidiosis. As expected, dimeric 8C3 ScFv antibody showed 3-folds higher antigen binding activity than monomer due to increased avidity. In addition, protien yields of dimer expression were 2-folds higher than monomer. Keywords: avidity, dimer, leucine zipper motif, single-chain Fv antibody 항체공학은주로재조합된항체단편의생산을목적으로하며주로 Fab, Fc, Fv, ScFv (single chain Fv) 항체등을생산한다 (20). 이중 ScFv 항체는직접적으로항원과결합하는부위인 VH와 VL의유전자를 linker peptide로연결하여만든재조합항체이다 (2, 7). 재조합 ScFv 항체제조기술은초기에는주로생쥐 ScFv 항체생산을목적으로하였으나, 최근에는닭의재조합 ScFv 항체를생산하는데에도성공적으로적용되고있다 (21, 23). 재조합 ScFv 항체의항원결합력을높이기위해서는항원과항체사이의친화력즉 affinity를높여주는방법도있으나, 이는항원결합력이높은 VH와 VL 유전자를찾아내어새로운 ScFv 항체를제조하는것으로많은시간과비용을요구하므로이에대한대안으로항원친화력즉 affinity는변하지 * For correspondence. E-mail: jkk7465@unitel.co.kr; Tel.: +82-55- 213-3485; Fax: +82-55-213-3480 않고항원과항체간의탐욕성즉 avidity를증가시키는방법을고려해볼수있다. Avidity를증가시키는방법으로항체의이량체화 (dimerization) 를통해항체와항원간의결합력을높여줄수있는데, 그방법으로는 ScFv 항체의 C-terminus에항체의 Fc 부분을도입함에의한이량체화 (18) 와항체의 hinge ( 경첩 ) 부분만을도입하여 miniantibody형태로이량체화하는방법 (11, 13, 18), 또는 Fc나 hinge 부분에서이황화결합을만들어주어이량체형성의결정적인역할을하는 cysteine 잔기를포함시켜인위적으로만든아주짧은 peptides를도입함 (4, 6, 15, 24) 으로이량체화를유도하는방법등이있으며이는모두공유결합인이황화결합을이용한다는공통점을가진다. 한편비공유결합을이용한 avidity 증가방법으로 leucine zipper motif를도입하는방법이보고되었다 (5, 9, 12). Leucine zipper motif는 yeast의 transcriptional activator인 GCN4로부터유래된 sequence로써 leucine 잔기가주기적으로나열되어
닭의재조합이량 ScFv 항체개발 329 지는 polypeptide 조각이며, 7개의 peptide간격으로주기적으로나열되어진이 leucine 잔기가 alpha helix를형성하도록하며주변의동일한 alpha helix간에비공유결합인소수성결합에의한이량체형성을가능하게한다 (5, 9, 12). 이전연구에서는조류에서 Coccidiosis를일으키는 Eimeria 속 (genus) 에속하는 Protozoa의 sporozoite가숙주세포에침투하는과정에관여하는 Conoid에특이적으로결합하는단일클론항체를분비하는 8C3 hybridoma로부터 variable heavy chain (VH) 과 variable light chain (VL) 유전자를 RT-PCR에의해클로닝하여 12개의아미노산으로이루어진 soluble linker (GSTSGKPSEGKG) 에의해연결된재조합 ScFv 항체를제조하여항원결합력을확인하였다 (10, 16). 본실험에서는이미만들어진단량체형태의 8C3 ScFv 항체에 amphipathic alpha helix가융합되도록 ScFv 항체의 C-terminus에 leucine zipper motif를도입하여, 이량체형태의재조합 8C3 ScFv 항체발현을 E. coli를숙주로하여시도하였다. 정제된이량체형태의재조합 8C3 ScFv 항체는 size exclusion chromatography에의해이량체임을확인하였고 ELISA 분석을이용하여단량체 8C3 ScFv 항체에비해 avidity 증가로인하여항원결합력이 3배정도증가됨을확인할수있었다. 또한 Westernblot 분석과 immunocytochemistry 등을통해서도이량체 8C3 ScFv 항체의항원결합력을재확인할수있었다. 재료및방법 Leucine zipper motif 를이용한이량체형태의발현벡터구축 Leucine zipper motif 유전자는 oligonucleotide를이용한유전자합성방법으로자체제작하였으며이를이전실험에서구축했던단량체형태의 8C3 ScFv 항체발현벡터에 NotI 제한효소를이용해클로닝하였다 (Fig. 1) (8, 16). 8C3 Fv 항체의 3 차구조모델링 항체 3차구조모델링을위한 web site인 WAM (http://anti body.bath.ac.kr/) 을이용하여 8C3 Fv 항체 (VH and VL complex) 의 3차구조모델링을시도하였다. 즉 8C3 Fv 항체의아미노 산배열을입력하면그와가장유사한아미노산배열을가지면서이미 3차구조가알려진항체를 RCSB PDB (http://www. rcsb.org/pdb/) 에서찾아내어이를주형으로하여 8C3 Fv 항체의항원결합부위인 complementarity determining region (CDR) 의 loop와항체의안정화역할을담당하는 framework (FR) 의 β-sheet의 3차구조를결정하였다. 예측된 3차구조는 PyMol (Delano Scientific, USA) 프로그램을이용하여형상화하여분석하였다. 이량체형태의 8C3 ScFv 항체발현및정제 ScFv 항체유전자를포함한발현벡터를 E. coli BMH71-18 에형질전환하였다 (8). 형질전환된 E. coli BMH71-18을 2XTY 액체배지에 100 μg/ml의농도가되도록 ampicillin (Sigma- Aldrich, USA) 과 1% (w/v) glucose를첨가하여 30 C에서 180 rpm으로밤새진탕배양하였고, 2,500 g로원심분리하여액체배지를제거후 2 TY 액체배지로모아진 E. coli를재현탁하여다시원심분리하였다. 재현탁되어진배양액에 100 μg/ml 의 ampicillin을다시넣어주고 0.25 mm IPTG를처리하여 25 C, 200 rpm에서 5.5 h 동안배양하였다. 배양이끝난세포배양액을원심분리하여상등액을제거하고, 수거된세포에차가운 TES buffer (0.5 M Sucrose, 0.1 mm EDTA and 200 mm Tris-Cl, ph 7.5) 를첨가하여 4 C에서 1 h 동안배양하였다. 다시위와같은방법으로원심분리하여수거된상등액은 PBS를이용해밤새투석하였다. 밤새투석된단백질샘플용액을 Ni-NTA agarose에결합시키고, Ni-NTA agarose 부피의 20배에해당하는 TN buffer (0.1 M Tris-Cl, 0.5 M NaCl, ph 7.5 and ph 8.0) 를이용하여세척하였다. 세척이끝난 Ni-NTA agarose에 250 mm immidazole을처리하여재조합단백질을용출해냈다. 또한 TES buffer 처리후수거된세포덩어리를초음파파쇄를위한 buffer (0.1 M Tris-Cl, 0.5 M NaCl, ph 7.5) 에재현탁한후초음파파쇄하여원심분리후상등액을수거하여위와같은방법으로정제하였다. 정제된단백질들을 2 SDS-PAGE buffer와섞어준후 100 C에서 5 min 끓여 15% SDS-PAGE gel에전기영동하고, 이를 0.25% Coomassie Brilliant Blue R-25 용액을이용하여 Fig. 1. Construction of dimeric 8C3 ScFv by addition of leucine zipper motif. The 8C3 dimeric ScFv is constructed from monomeric ScFv expression vector derived from puc119 containing a 5 -PelB leader sequence and 3 -hexahistidine tag by insertion of leucine zipper motif at C-terminus of ScFv.
330 Park et al. (A) (B) Fig. 2. 3D modeling of chicken 8C3 Fv. (A) Side view. (B) Top view. Coloered region represent each CDR (HCDR 1: red, HCDR2: green, HCDR3: blue, LCDR 1: yellow, LCDR 2: magenta and LCDR 3: Cyan) 염색하였다. 정제된 ScFv 항체단백질은 Bradford protein assay (Thermo scientific, USA) 를이용해정량하고실험에이용하였다. Size exclusion chromatography 정제된재조합항체들은 Superdex G-75 gel (GE Health care, UK) 이채워져 25 C에서 10배볼륨의 PBS (ph 7.4) 로 equilibration 하였고, column에 1-1.5 ml/min의속도로통과시켜각분획당 1 ml씩모아주었다 (Model 2128, BioRad, USA). 이렇게모아진분획들을위에서언급한방법대로 SDS- PAGE를통해정제하여단백질의분자량을측정한다. 분자량의비교를위해크기가알려진단백질인 bovine serum albumin (67 kda) 과 carbonic anhydrase (30 kda) 을같은조건에서 column을통과시켜분획하였다. Enzyme linked immunosorbent assay (ELISA) 4 C에서 2 μg의 Eimeria tenella antigen이고정된 plate (Maxisorp, Nunc, Denmark) 는 PBS에 0.1% 의 Tween 20을첨가한 PBS-T buffer로세번세척하였고, 여기에 skim milk 를 2% 포함한 PBS-T buffer를 blocking에이용하였다. Blocking 이끝난후정제된 ScFv 항체를포함한 blocking buffer를처리하였고, PBS-T를이용한 3번의세척후 horseradish peroxidase가 conjugated polyhistidine monoclonal antibody (Sigma- Aldrich) 를처리하여 ABTS (Sigma-Aldrich) 와과산화수소로발색하였다. 발색된 plate는 Microplate reader (EXPERT 96, Asys, Austria) 를이용해 405 nm 파장에서흡광도를측정하였다. Western blot 수용성의 Eimeria tenella antigen을전기영동방법으로 SDS- PAGE하고 PVDF membrane (Millipore, USA) 으로 transfer 하였다. 그리고 transfer 된 membrane을 2% (w/v) skim milk 를포함한 PBS-T buffer를이용하여실온에서 1 h 동안 blocking한다음, PBS-T로 2회세척하였다. Membrane을이량체형태의 8C3 ScFv 항체 10 μg을포함하는 PBS-T blocking buffer에담가서실온에서 1 h 동안 incubation한후 PBS-T로 3 회세척하였다. 발색을위해 2차항체인 peroxidase-conjugated polyhistidine monoclonal antibody (Sigma-Aldrich) 를 1:2,000 (v/v) 으로희석해서 membrane에처리하여실온에서 1 h 동안 incubation하였다. PBS-T washing buffer로 5회세척한다음, 3,3-diaminobenzidine (Sigma-Aldrich) 를이용해발색시켜이량체 8C3 ScFv 항체의항원결합력을재확인하였다. Immunocytochemistry PBS에섞어둔 Eimeria tenella sporozoites을실리콘처리된 glass slide (Dako, Japan) 위에얹어자연건조시키고, 차가운 methanol에 2분동안담궈고정시킨후 PBS를이용해 3 번세척하였다. Slides에고정된 sporozoites에 2 μg의이량체 8C3 ScFv 항체를처리하여습윤상자에넣고실온에서 1 h 동안 incubation하였다. Slides를 PBS로 3회세척후 PBS에 mouse anti-polyhistidine tag antibody를 1:1,000 (v/v) 로희석해준항체용액을 100 μl 처리하여 1 h 동안 incubation한다음 PBS를이용해 3회반복세척해주고, 100 μl의 1:1,000 (v/v) 으로희석된 fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG F(ab) (Sigma-Aldrich) 를처리하여 1 h 동안 incubation 하였다. 최종적으로 Slides를 PBS로 3회반복세척하고공초점주사현미경 (Nikon, Japan) 을사용하여 sporozoites 항원의이량체 8C3 ScFv 항체에의한형광염색정도를관찰하였다. 결과및고찰이량체 8C3 ScFv 항체발현벡터는합성된 leucine zipper motif 유전자를클로닝하여제한효소 NotI에의해단량체의 C-terminus에삽입하여 8C3 ScFv/Leucine zipper motif형태로구축되었다 (Fig. 1). 즉, leucine zipper motif 유전자의 upstream에 ScFv 항체유전자를 SfiI과 NotI의제한효소를이용하여클로닝하면자동적으로 leucine zipper motif와융합된발현벡터가구축되도록고안되었다 (Fig. 1). 예측된 8C3 Fv
닭의재조합이량 ScFv 항체개발 331 항체 3차구조를보면 VH 및 VL의각각세 CDR 부위가고유한 loop 구조를나타내며 (Fig. 2A) 6개각각의 CDR loop가다시 (Fig. 2B) 항원결합부위인 hydrophobic pocket을형성하여항원과특이적으로결합할수있게된다. 구축된 8C3 ScFv/Leucine zipper motif 발현벡터를이용하여 E. coli를형질전환하고, IPTG를이용하여단백질발현을유도한후 Ni-NTA affinity chromatography를이용하여이량체 8C3 ScFv 항체를정제하였다. SDS-PAGE에서이량체분자량을분석해본결과 leucine zipper domain이첨가된관계로단량체크기인약 30 kda 보다큰약 38 kda으로확인되었다 (Fig. 3). 그리고, SDS-PAGE 분석에의해서는비공유결합에의한이량체형성을결정할수없으므로비공유결합의일종인소수성결합의한이량체형성을확인하기위해정제된 ScFv 항체단백질을 size exclusion chromatography를통해분획하였고, 이량체및단량체 8C3 ScFv 항체단백질분획의용출위치를결정하기위해이미분자량이알려진 bovine serum albumin (67 kda) 와 carbonic anhydrase (30 kda) 을동일조건에서분획하였다. 이량체 8C3 ScFv 항체는 67 kda 크기의 BSA와같은분획에서주로용출되었고, 단량체 ScFv 항체가분획되는 pattern은 30 kda 크기의 carbonic anhydrase와유사하였다 (Fig. 4). 이결과는정제된이량체 8C3 ScFv 항체가 leucine zipper motif간의비공유결합인소수성결합에의해성공적으로이량체를형성하는것을보여주고있다. 또한발현된단백질의회수율 (yield) 을분석한결과이량체형태의 Fig. 3. SDS-PAGE analysis of dimeric 8C3 ScFv. The monomeric 8C3 ScFv (Lane 1) and dimeric 8C3 ScFv (Lane 2) expressed in E. coli were purified and analyzed in 15% polyacrylamide gel under reducing condition. ScFv 항체는 leucine zipper motif를구성하는수용성아미노산잔기에의한수용성증가효과로단량체일때의 0.21 mg/l 에비해 2배정도발현율이증가된 0.42 mg/l로발현되었다 (Fig. 5). 수용성증가효과를확인하기위해 leucine zipper motif의소수성정도를 Web-based 프로그램 (http://web.expasy. org/protscale/) 을이용하여분석한결과, leucine zipper motif Fig. 4. Size exclusion chromatography. After gel filtration through Superdex G-75 column, SDS-PAGE showed recombinant dimeric 8C3 ScFv has similar elution patterns with bovine serum albumin (BSA). BSA and carbonic anhydrase were molecular weight standard as 67 kda and 30 kda, respectively.
332 Park et al. Recombinant Protein Fig. 5. Protein expression yield. Expressed and purified protein yield of recombinant dimeric 8C3 ScFv is 2 fold higher than monomer. 는그자체로매우높은친수성을보여주었다. 그리고 leucine zipper motif는소수성과친수성을동시에갖는특징 (amphipathic nature) 을띄고있다. 이는기존의단량체형태의 ScFv 항체에 leucine zipper motif가도입되어친수성이높은영역이늘어남으로상대적으로단량체에비해친수성이커지게될뿐만아니라 leucine zipper motif상의소수성영역마저도소수성결합시 alpha helix들간의결합에의해소수성영역이내부적으로서로를감싸는형태가되어외부로나타내는친수성비율을더욱높임으로써단백질회수율 (yield) 이 2배정도 증가하는것으로생각되어진다. 이량체 8C3 ScFv 항체의주된기능인항원결합능력을측정하고단량체의결합력과비교하기위해 ELISA 분석을시도한결과이량체형태의 8C3 ScFv 항체가단량체에비해대략 3배정도의항원결합능력을가지고있음을확인할수있었다 (Fig. 6A). 그리고 Western blot 분석결과에서는이량체 8C3 ScFv 항체가 55 kda과 60 kda의 sporozoites 항원을특이적으로탐지하는것을확인할수있었다 (Fig. 6B). 이량체 8C3 ScFv 항체가 Figure 6B와같은다특이성 (multispecificity) 을나타내는것은이량체 8C3 ScFv 항체가직접결합하는부위 (peptides: epitope) 가 55 kda과 60 kda항원에공통적으로존재하거나, 또는인지하는항원이 55 kda과 60 kda 항원에존재하는구조적으로유사성이높은당 (carbohydrate) 일가능성을암시해주고있다. 또한 Immunocytochemistry를통해이량체 8C3 ScFv 항체가 Eimeria tenella sporozoites의 conoid 구조에특이적으로결합하여형광을나타내는것으로이량체 8C3 ScFv 항체의항원결합능력을재확인할수있었다 (Fig. 7). 이러한결과들로이미보고된것처럼 (5, 11, 13) leucine zipper motif의도입이소수성결합에의한이량체를형성하고, 이로인한항원결합부위증가 (bivalency) 로 avidity 가증가되어항원결합력이단량체에비해 3배증가된것을알수있었다. 즉다량체형성 (multimerization) 으로항원결합부위를증가시켜 avidity를향상시켜전체항원결합능력을증가시키는방법을제시해주고있다. 자연계의모든항체는 Y 형태로써 2개의항원결합부위를갖는다. 그러나재조합 ScFv 항체는단량체형태로써 1개의항원결합부위만을가지므로 Y형태의모항체에비해항원결 (A) (B) Fig. 6. Antigen binding activities of dimeric 8C3 ScFv by ELISA and Western blot. (A) Two micrograms of soluble Eimeria tenella sporozoites antigens were coated on 96 well plate and same amounts of monomeric and dimeric 8C3 ScFv were treated as primary antibody. The dimeric ScFv has 3 folds of antigen binding activity of monomer. (B) Eimeria tenella sporozoites antigens were resolved by SDS-PAGE and transferred to PVDF membrane. 10 μg of dimeric ScFv was treated to membrane as primary antibody. Membrane was developed with 3,3-diaminobenzidine/H 2O 2. After development, two specific bands of sporozoites antigen were detected as 55 kda and 60 kda.
닭의재조합이량 ScFv 항체개발 333 단량체형태의 (monomer) single chain Fv 항체에비해항원결합력 (Avidity) 이증가될것이다. 이개념에기초하여이전연구에서제조된단량체형태인닭 single chain Fv 항체인 8C3 ScFv 항체의 C terminus에 leucine zipper motif를도입하여이량체형태의 8C3 ScFv 항체를개발하였다. 이량체 8C3 ScFv 항체는가금류의대표적인기생충질병인 coccidiosis를유발하는 Eimerian sporozoite에특이적으로결합하는기능을나타내었다. 또한이량체 8C3 ScFv 항체는 avidity 증가로인하여단량체에비해항원결합력이약 3배증가됨을확인할수있었으며단백질회수율또한 2배증가되는부수적인효과를얻을수있었다. Fig. 7. Immunocytochemistry. Eimeria tenella sporozoites were fixed on siliconized glass slide and dimeric ScFv was treated on the slide. After FITC conjugated secondary antibody treatment and washing, slide was photographed using confocal laser scanning microscope (Nikon, Japan). 합력즉 avidity가낮은약점을가지고있으므로이를보완하기위해, leucine zipper motif나 Fc region, hinge region, cysteine peptide등의이량체형성유도인자를첨가하여이량체 ScFv 항체제조를유도하는다양한방법이보고되었다 (1, 3, 5, 11, 14, 22). 이러한형태의이량체 ScFv 항체는 2개의항원결합부위를유지하게되어모항체와동일한항원결합력을유지할수있는잇점외에도이량체형성유도인자의친수성에의한단백질의발현및회수율, 단량체 ScFv 항체의구조적불안정성을보완해주는추가적인효과를얻을수있다. 또한항원특이성이다른 2개의단량체 ScFv 항체를위의방법으로이량체화하면전혀다른 2가지항원을동시에인지하는이중특이성 (bispecificity) 을갖는재조합 ScFv 항체개발도이미보고되었다 (5, 11, 17, 19). 본연구의내용을요약하면가금류의대표적질병인 Coccidiosis를일으키는 Eimerian sporozoite에특이적으로결합하는닭의재조합이량체 8C3 ScFv 항체를제조하여 avidity를증가시킴으로써단량체 ScFv 항체의단점을극복하였으며또한 Eimerian sporozoite항원의성공적인탐지및염색결과는이량체 8C3 ScFv 항체의가금류 Coccidiosis 질병의진단및치료제로사용할수있는가능성을제시해주고있다. 적요 Leucine zipper motif는여러개의주기적인 leucine 잔기로구성되어 amphipathic alpha helix형태의구조를나타내며소수성결합에의해이량체를형성한다. 이 leucine zipper motif를 single chain Fv 항체의 C terminus에도입하면 leucine zipper motif의소수성결합에의해 amphipathic alpha helix의이량체가형성되면서융합된 single chain Fv 항체의이량체 (Dimer) 형성또한유도할수있다. 이량체형태의 single chain Fv 항체는 2개의항원결합부위를갖게되므로 감사의말 본연구는 2011년도창원대학교교내공모과제지원에의해서수행되었으며이에감사드립니다. 참고문헌 1. Albrecht, H., G.L. Denardo, and S.J. Denardo. 2006. Monospecific bivalent scfv-sh: effects of linker length and location of an engineered cysteine on production, antigen binding activity and free SH accessibility. J. Immunol. Methods 310, 100-116. 2. Bird, R.E., K.D. Hardman, J.W. Jacobson, S. Johnson, B.M. Kaufman, S.M. Lee, T. Lee, S.H. Pope, G.S. Riordan, and M. Whitlow. 1988. Single-chain antigen-binding proteins. Science 242, 423-426. 3. Brinkmann, U., M. Gallo, E. Brinkmann, S. Kunwar, and I. Pastan. 1993. A recombinant immunotoxin that is active on prostate cancer cells and that is composed of the Fv region of monoclonal antibody PR1 and a truncated form of Pseudomonas exotoxin. Proc. Natl. Acad. Sci. USA 90, 547-551. 4. Brinkmann, U., Y. Reiter, S.H. Jung, B. Lee, and I. Pastan. 1993. A recombinant immunotoxin containing a disulfidestabilized Fv fragment. Proc. Natl. Acad. Sci. USA 90, 7538-7542. 5. de Kruif, J. and T. Logtenberg. 1996. Leucine zipper dimerized bivalent and bispecific scfv antibodies from a semi-synthetic antibody phage display library. J. Biol. Chem. 271, 7630-7634. 6. Glockshuber, R., M. Malia, I. Pfitzinger, and A. Pluckthun. 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29, 1362-1367. 7. Huston, J.S., D. Levinson, M. Mudgett-Hunter, M.S. Tai, J. Novotny, M.N. Margolies, R.J. Ridge, R.E. Bruccoleri, E. Haber, R. Crea, and et al. 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879-5883. 8. Kim, J.K., M.F. Tsen, V. Ghetie, and E.S. Ward. 1994. Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis. Eur. J. Immunol. 24, 542-548. 9. Landschulz, W.H., P.F. Johnson, and S.L. McKnight. 1988. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759-1764. 10. Lawn, A.M. and M.E. Rose. 1982. Mucosal transport of Eimeria tenella in the cecum of the chicken. J. Parasitol. 68, 1117-23. 11. Muller, K.M., K.M. Arndt, and A. Pluckthun. 1998. A dimeric
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