Protein Expression and Purification 91 (2013) 20–29

Contents lists available at SciVerse ScienceDirect

Protein Expression and Purification

journal homepage: www.elsevier .com/ locate /yprep

Expression and characterization of an enantioselective antigen-bindingfragment directed against a-amino acids

1046-5928/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.pep.2013.06.010

⇑ Corresponding author. Fax: +1 815 753 4802.E-mail address: hhofst@niu.edu (H. Hofstetter).

1 Current address: Indiana University School of Dentistry, Indianapolis, IN 46202,USA.

2 Abbreviations used: IgG, Immunoglobulin G; CDRs, complementarity detregions; NCBI, National Center for Biotechnology Information; HRP, hoperoxidase; DMAP, dimethylaminopyridine; DSC, disuccinimidyl carbonatriethylamine; BLAST, Basic Local Alignment Search Tool; FR1, framework rePCR, polymerase chain reaction; C1, constant region 1.

Pierre P. Eleniste 1, Heike Hofstetter ⇑, Oliver HofstetterDepartment of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115-2862, USA

a r t i c l e i n f o

Article history:Received 30 January 2013and in revised form 19 June 2013Available online 1 July 2013

Keywords:Enantioselective antibody fragmentStereoselective FabExpressionOverlap extension PCRMouse antibody constant regions

a b s t r a c t

This work describes the design and expression of a stereoselective Fab that possesses binding propertiescomparable to those displayed by the parent monoclonal antibody. Utilizing mRNA from hybridomaclones that secrete a stereoselective anti-L-amino acid antibody, a corresponding biotechnologically pro-duced Fab was generated. For that, appropriate primers were designed based on extensive literature anddatabank searches. Using these primers in PCR resulted in successful amplification of the VH, VL, CL andCH1 gene fragments. Overlap PCR was utilized to combine the VH and CH1 sequences and the VL and CL

sequences, respectively, to obtain the genes encoding the HC and LC fragments. These sequences wereseparately cloned into the pEXP5-CT/TOPO expression vector and used for transfection of BL21(DE3) cells.Separate expression of the two chains, followed by assembly in a refolding buffer, yielded an Fab that wasdemonstrated to bind to L-amino acids but not to recognize the corresponding D-enantiomers.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Due to their unsurpassed binding properties, immunoglobulinsare probably the most widely used affinity capture reagents. Fordecades, both polyclonal and monoclonal antibodies have been em-ployed in, e.g., biochemical research, diagnostics, and therapeuticapplications [1–3]. More recently, advances in molecular biologicalmethods have opened up new opportunities for the biotechnologi-cal production and targeted manipulation of antibody-fragments,which have quickly gained even greater popularity for, e.g., thedevelopment of novel antibody-based drugs [4,5].

As early as in 1928, Karl Landsteiner recognized that appropri-ately raised antibodies may exhibit a high degree of stereoselectiv-ity. However, this discriminatory potential appears to have largelybeen neglected by the scientific community. One noteworthyexception is the field of catalytic antibodies where, starting in the1980s, antibodies have been produced that can catalyze the conver-sion of suitable substrates with stereo- and regioselectivity [6–8].Only few applications have utilized antibodies that stereoselective-ly bind to molecules in their ground state (as opposed to moleculesin the transition-state, as is the case with catalytic antibodies). Suchantibodies have mostly been used in radioimmunoassays for thedetection of chiral drugs and their metabolites, respectively [9].Only within the last fifteen years, stereoselective antibodies have

become more popular tools for the analysis of chiral molecules[10]. For example, it has been demonstrated that stereoselectiveantibodies can be utilized for the ultra-sensitive detection of enan-tiomers in immunoassays [11,12] and sensors, [13–19] as well asfor chiral separation in chromatography [20–28]. With few excep-tions, [24–26] these studies typically utilized antibodies that hadbeen produced by classical immunological techniques and, thus, in-volved immunization of, e.g., rabbits or mice.

Since the biotechnological production of antibodies promises toreduce the need for laboratory animals, it is intriguing to use molec-ular biological techniques also for the generation of stereoselectiveantibody fragments. In addition, the availability of plasmids encod-ing the information for such antibodies not only opens the door totheir targeted manipulation and mass production, but also facili-tates further biophysical and structural biological characterization.

Immunoglobulin G (IgG)2 molecules are glycoproteins (MW �150 kDa) composed of four polypeptide chains, namely two lightchains (VL–CL) of about 25 kDa each, and two heavy chains(VH–CH1–CH2–CH3) of ca. 50 kDa each. The light and heavy chainsare held together by non-covalent bonds and disulfide bridges be-tween the CL and CH1 domains. An Fab fragment represents theantigen-binding arm of an antibody; it is composed of a light chain,LC, which consists of the VL and CL domains, and a heavy chain, HC,which comprises the VH and CH1 domains. Papain, a nonspecific

erminingrseradishte; TEA,

gion one;

P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29 21

thiol-endopeptidase, can be used to enzymatically cleave whole IgGmolecules at the hinge region to yield two identical Fab fragmentsand one Fc fragment [29–33].

The Fab fragments, which contain the complementarity deter-mining regions (CDRs), are known to be more stable than molecu-lar biologically produced single chain Fv (scFv) fragments [34,35].It is also known that Fab exhibit binding properties similar to orindistinguishable from those of their whole parent antibody[36,37]. Due to their small size, rapid blood clearance, and lowimmunogenicity, Fab have been widely utilized in diagnostic,therapeutic, and research applications [38,39]. For example, Fabfragments have been employed in the treatment of digoxin inges-tion, [40] snake bites, [41] Entamoeba histolytica, [42] rabies, [43]thrombosis, and tumors [44].

Here, we describe the construction and expression of a stereo-selective Fab fragment in Escherichia coli using the genetic materialof hybridoma cells that produce an antibody that stereoselectivelybinds to the L-enantiomers of a-amino acids but not to the corre-sponding D-enantiomers [28,45,46]. Similar parent antibodies havesuccessfully been used for enantiomer detection and separation ina variety of analytical techniques [11–17,20–23,27,28,45,46].

Experimental

Production and characterization of the parent antibody

The monoclonal anti-L-amino acid antibody 80.2 (anti-L-AA80.2) was produced with permission of the Institutional AnimalCare and Use Committee at Northern Illinois University (ORC#292) following previously published procedures [45,46].

Antibody stereoselectivity was first verified in noncompetitiveELISAs on 96-well microtiter plates (Nunc, Rochester, NY) usingthree different solid-phase coatings, namely either underivatizedbovine serum albumine (BSA), or the conjugates of BSA andp-amino-D- or -L-phenylalanine, prepared by diazotization.Competitive ELISAs were performed utilizing the BSA-conjugateof p-amino-L-phenylalanine as coating and the pure enantiomersof various a-amino acids as inhibitors.

Sequence analysis of monoclonal antibody anti-L-AA 80.2

The mRNA of 108 hybridoma cells producing anti-L-AA 80.2 wasisolated on an oligo(dT) spin column (Qiagen, Valencia, CA) andconverted into cDNA in a first-strand reaction with reverse trans-criptase using the primer first-strand mix kit (Amersham Pharma-cia Biotech, Little Chalfont, England). For the amplification ofantibody heavy and light chain DNA (�340 and �325 bp, respec-tively), the degenerate primers described by Wang et al. [47] wereused. Heavy and light chain DNA was ligated into the plasmidpCR4.1 for transfection into E. coli Top 10 (Invitrogen, Carlsbad,CA). Sequencing reactions were carried out by Dr. Scott Grayburnat the Core DNA Synthesis and Sequencing Facility at Northern Illi-nois University using a Beckman CEQ8000XL Capillary System(Beckman Coulter; Fullerton, CA).

The nucleotide sequences were analyzed using Sequencher, [48]and converted into the corresponding amino acid sequences usingthe translation tool on the ExPASY Server [49]. The program BasicLocal Alignment Search Tool (BLAST; Ref. [50]) was utilized toverify the origin of the sequences by comparing them with se-quences in the National Center for Biotechnology Information’s(NCBI) non-redundant (nr) database. The sequences were alignedwith mouse heavy or light chain variable regions compiled in theKabat database [51–53] to determine positions of the frameworkand hypervariable regions.

Production of stereoselective antibody fragments by molecularbiological techniques

Amplification of VH and VL fragmentsBased on sequence analysis of the monoclonal antibody 80.2,

two sets of primers, 80.2VH_fw (50-GAGGTCCAGCTGCAACAAT-CAGG-30) and 80.2VH_rev (50-GAGAGACATGACCAGAGTCCCTTGG-30) for VH, and 80.2VL_fw (50-GACATTGTGCTGACCCAATCTCCA-30)and 80.2VL_rev (50-GTTTCGCTCCAGCTTGGTCCCA) for VL were de-signed and used to amplify the variable sequences of heavy andlight chains from cDNA by PCR. Reaction mixtures were composedof 5 lL of 10� Taq buffer containing Mg+2 (Eppendorf, Hamburg,Germany), 1 lL of 10 mM deoxynucleotide triphosphates (Prome-ga, Madison, WI) at a final concentration of 0.2 mM, 1 lL of for-ward and reverse primers at a final concentration of 0.1–0.2 lM,1–150 ng/lL template DNA, 0.4–1 lL (corresponds to 0.5–2.5units) of Taq DNA polymerase (Eppendorf, Hamburg, Germany),and nuclease-free water to yield a total volume of 50 lL. The ther-mocycler program was as follows: 1 cycle at 94 �C for 3 min, fol-lowed by 30 cycles (94 �C for 1 min, 55 �C for 1 min, and 72 �Cfor 2 min) and a final cycle of 10 min at 72 �C. Amplified DNAwas analyzed in agarose gels and purified using the Wizard SVGel and PCR Clean-Up System (Promega, Madison, WI).

Construction of the mouse heavy constant chain (CH1) and the mousekappa constant chain (CL)

In order to identify appropriate primers for the amplification ofthe constant regions, several kappa light chain constant regions(CL) and heavy chain constant regions from different sources werecompared and evaluated. For the production of the CL domain, thesequence MOPC 21 # 8 from the Kabat database [53] was used fordesigning the forward and reverse primers: CL_fw (50-GCTGATGCTGCACCAACTGTATCCATCTTCCCACC-30) and CL_rev (50-ACACTCATTCCTGTTGAAGCTCTTGACAATGG-30). The CH1 nucleotide sequencefrom BC024405.1 (from BLAST) was selected as template for thedesign of the primers CH1_fw_constant (50-CCAAAACGACACCCCCATCTGTCTATCCACT-30) and CH1_rev_constant (50-ACCACAATCCCTGGGCACAATTTTCTTGTCCACC-30), which were used to amplify theCH1 sequence from 80.2 cDNA.

The PCR conditions for the amplification of the constant regionswere the same as described above for the variable regions.

Assembly of the 80.2 HC and LC fragments

To allow generation of the whole 80.2 HC sequence by PCR, thetwo fragments, 80.2 VH and 80.2 CH1, should ideally overlap by 15–35 bp. The forward primer, CHextension80.2VH: 50-CTGGGGCCAAGGGACTCTGGTCATGTCTCTCGCCAAAACGACACCCCCATCTGTCT-ATCCACT-30, was designed, which contains the antisense of the 80.2VH 30 end at its 50 end (represented by the underlined nucleotides).

Using the above described PCR conditions, an overlap with 80.2VH was created at the 30 start of 80.2 CH1 resulting in an extended80.2 CH1.

For the 80.2 LC sequence, the primer CL.o/v.80.2VL.: 50-TGGGACCAAGCTGGAGCGAAACGCTGATGCTGCACCAACTGTATCCATCTTC-30

was designed with a 20 bp overhang from the 30end of the 80.2 VL

complement region. This produced an extended 80.2 CL sequence.Overlap extension PCR was then carried out for the creation of

the heavy chain (HC) and light chain (LC) fragments, using VH

and extended CH1 and VL and extended CL sequences, respectively.Primers utilized for HC were 80.2VH_fw and CH1_rev_constant,while the primers for LC were 80.2VL_fw and CL_rev_constant.PCR was performed using 5 lL of 10� Taq buffer, 1 lL of the appro-priate forward and reverse primers, 5 lL of appropriate DNA tem-plates, 2.5 lL dNTPs, 5 lL MgCl2 (at 25 mM), 0.7–1.0 lL (0.5–2.5

Table 1Effect of the induction time on protein expression yields.

Protein Time of induction (h) Yield (mg/1 L culture)

80.2 HC 0 5080.2 LC 0 5780.2 HC 4 17880.2 LC 4 16680.2 HC 8 13080.2 LC 8 12780.2 HC 16 10080.2 LC 16 100

22 P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29

units) Taq DNA polymerase, and sterile water to obtain a final vol-ume of 50 lL. PCR conditions were as follows: 1 cycle at 94 �C for3 min, followed by 30 cycles (94 �C for 1 min, 55 �C for 2 min, and72 �C for 2 min) and a final extension for 10 min at 72 �C. PCR prod-ucts were purified from agarose gels using the Wizard SV Gel andPCR Clean-Up System.

Expression of the Fab protein

HC and LC fragment DNA sequences, respectively, were ligatedinto the pEXP5-CT/TOPO cloning vector using a TOPO cloning reac-tion. DNA was analyzed in agarose gels and purified using the Wiz-ard SV Gel and PCR Clean-Up System prior to electroporation andtransformation into E. coli BL21 DE3. The 80.2 HC and 80.2 LC anti-body proteins were expressed in separate cultures. Cells in LB(5 mL) containing ampicillin (100 lg/mL) were inoculated withboth vectors and 1 L cultures were grown overnight (37 �C;250 rpm) to an OD600 of 0.5–0.7. Each cell culture was then in-duced with IPTG (1 mM final concentration) and incubated for4 h (37 �C, 250 rpm).

The cells were harvested by centrifugation at 3200 rpm for20 min at 4 �C. Extraction of the recombinant protein from theBL21(DE3) E. coli cells was performed using the B-PER II BacterialProtein Extraction Reagent (Thermo Fisher, Rockford, IL) accordingto the manufacturer’s instructions. Other lysis buffers were inves-tigated but did not show any improvement in yield. The totalamount of secreted protein in bacterial culture supernatants wasvery low. To increase protein yield, cell pellets from 1 L bacterialculture were resuspended with 30–50 mL B-PER (between 15and 25 g wet cells were obtained from a 1 L culture). After incuba-tion for 15 min cell debris was removed by centrifugation at13,000 rpm for 5 min. Whole cell lysates from 1 L culture werethen suspended in 50 mL of unfolding buffer consisting of 6 Mguanidine-HCl, 50 mM NaH2PO4, 300 mM NaCl, 2 mM reducedglutathione (GSH), and 0.2 mM oxidized glutathione (GSSG), pH7.4, and stirred for 24–36 h at 4 �C. Centrifugation (15 min at8000 rpm) was used to remove any insoluble material [54].

Different induction times (0–16 h) were investigated to opti-mize protein expression. For this experiment, both fragments wereHis-tagged. Protein samples containing His-tagged HC and LC,respectively, were dialyzed against PBS and purified on Talon Me-tal Affinity resin (Clontech Laboratories, Mountain View, CA).Depending on the amount of protein to be purified, column bedvolumes were either 5 or 50 mL with a capacity of 3 mg of proteinper 2 mL of gel. In brief, the talon resin was resuspended, trans-ferred to a sterile tube and centrifuged at 700g for 2 min to pelletthe resin. The supernatant was removed and discarded. The resinwas then briefly mixed with 10 bed volumes of equilibration/washbuffer (50 mM Na3PO4, 300 mM NaCl, pH 7.0) and pre-equlibratedbefore recentrifugation at 700g for 2 min. The equilibration/washstep was repeated two more times. Then, the protein sample wasadded to the resin and the mixture was agitated at room temper-ature for 20 min on a platform shaker to allow the His-tagged pro-tein to bind to the resin. The mixture was centrifuged at 700g for5 min and the supernatant was carefully removed. The pellet wasresuspended in 10–20 bed volumes of equilibration/wash bufferand gently agitated at room temperature for 10 min on a platformshaker. The suspension was then centrifuged at 700g for 5 min andthe supernatant removed and discarded. This washing step was re-peated two more times. Then, one bed volume of equilibration/wash buffer was added and the resin resuspended by vortexing.The resin was transferred to a gravity flow colum with an end-cap in place and allowed to settle out of suspension. The endcapwas removed and the buffer allowed to drain until it reached thetop of the resin bed. The column was washed once with 5 bed vol-umes of equilibration/wash buffer. His-tagged protein was eluted

by adding 5 bed volmes of imidazole elution buffer (50 mM Na3-

PO4, 300 mM NaCl and 150 mM imidazole, pH 7.0) to the column.Eluate was collected in 500 lL fractions. The protein content of allfractions was determined by measuring the UV absorbance at280 nm. Protein containing fractions were pooled and concen-trated using Amicon Pro 10 K centrifugal concentrators (EMD Mil-lipore, Billerica, MA). Protein concentrations were determinedusing a NanoDrop� ND-1000 UV/Vis (Wilmington, DE, USA;Table 1). Spectra were recorded to ensure that no contaminationfrom DNA/RNA was observed.

For the assembly of the Fab, expression was carried out asdescribed above but the LC fragment was not His-tagged to allowfor purification of assembled Fabs. Solutions in unfolding buffercontaining His-tagged HC and native LC fragments were mixed ina 1:1 ratio in order to allow heavy and light chains to associate.The resulting solution was diluted 1:10 with refolding buffer con-taining 20 mM Tris–HCl (pH 7.4), 1.0 mM EDTA, 2.0 mM GSH, and0.2 mM GSSG. The mixture was gently stirred for 2 days at 4 �C tofacilitate correct folding of protein. The protein solution was thendialyzed overnight against PBS (pH 7.4) at 4 �C.

Purification of His-tagged Fab was carried out on Talon MetalAffinity resin as described above for LC and HC fragments andsamples were concentrated using Amicon Centriplus 30 K centrifu-gal concentrators (EMD Millipore, Billerica, MA). Purified sampleFab fragments were analyzed by Western Blot and protein concen-trations were determined using a NanoDrop� ND-1000 UV/VisSpectrophotometer.

Characterization of Fab fragments

Expressed Fabs were tested by Dot blot and ELISA. The percentageof active protein was estimated utilizing affinity chromatography.

Dot blot: 2 lL samples (at 1 mg/mL) were applied to a nitrocel-lulose membrane (Protran� pure nitrocellulose membrane, Schlei-cher & Schuell Inc., Keene, NH). Dots were dried at roomtemperature for about 1 h. After washing the membrane severaltimes with PBS containing 0.05% (v/v) Tween 20 (PBS/Tween 20),unoccupied sites on the membrane were blocked by immersing itin PBS/Tween 20 containing 1% gelatin for 1 h at room tempera-ture. The membrane was then washed multiple times with PBS/Tween 20, followed by incubation in a 1/5000 dilution of horserad-ish peroxidase (HRP) labeled-secondary antibody (Jackson Immu-noResearch Laboratories, West Grove, PA) in PBS (1 h at roomtemperature). After washing the membrane again several timeswith PBS/Tween 20, tetramethyl benzidine (TBS; Endogen, MA,USA) in the presence of 13 lL of 50% hydrogen peroxide was addedas substrate for color development. The blotted membrane wasrinsed in water to stop the reaction.

Affinity chromatography: A stationary phase was produced thatcomprised an azo-structure analogous to the one contained in thehapten-carrier conjugate that was used in the production of theparent antibody. For that, 14 g Sepharose CL4B (wet weight) werewashed successively with 1:3 (v/v) acetone:water, 1:1 (v/v)

P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29 23

acetone:water, 3:1 (v/v) acetone:water, and dry acetone. The sup-port material was then activated with a solution of 1.65 g ofdisuccinimidyl carbonate (DSC) and 1.35 g of dimethylaminopyri-dine (DMAP) dissolved in 20 mL of DMSO following the procedureby Wilchek and Miron [55]. The reaction was allowed to proceedovernight at 4 �C under gentle agitation. The activated stationaryphase was successively washed with dry acetone, 5% acetic acidin 1,4, dioxane, methanol and finally isopropyl alcohol. The numberof active groups was determined spectrophotometrically at261 nm using a 1/10 dilution of a 4 mg aliquot of the Sepharosethat had been hydrolyzed using 1 mL of 0.25 M ammoniumhydroxide. Next, 200 mg of tyramine in 30 mL of PBS was reactedwith the activated support material for 4 h at 4 �C. The stationaryphase was then washed successively with PBS, 100 mM Tris buffer(pH 7.0), PBS, 1 M NaCl and again PBS. Then, 20 mgp-amino-L-phenylalanine was dissolved in 1 mL of HCl, and anequimolar amount of sodium nitrate was added. After 10 min,the reaction mixture was slowly added to the suspension of sta-tionary phase. Production of an azo-structure was obvious from achange in color to orange-red. The pH as adjusted to 8.5 with0.1 M NaOH and the reaction allowed to proceed overnight at4 �C. The prepared stationary phase was washed extensively withPBS, NaCl, triethylamine (TEA), citric acid, HCl and guanidine-HCl.Following each washing, the stationary phase was rinsed withwater. 4.2 mg of the Fab in PBS was incubated with the affinity sta-tionary phase for 2 h at room temperature and then poured into a20 mL glas column. The flow-through was collected and the col-umn was washed first with PBS containing 1 M NaCl and then withPBS until no UV-absorbance at 280 nm was detected. Antibody waseluted utilizing 10 mM TEA (pH 12), and 1 mL fractions were col-lected and immediately neutralized with 27 lL of 100 mM citricacid. Fractions with significant protein levels (as determined bymeasuring the UV-absorbance at 280 nm) were combined and dia-lyzed against PBS. Purified protein solutions were concentratedusing Amicon Pro 10 K centrifugal concentrators (EMD Millipore,Billerica, MA). Protein concentrations were determined using aNanoDrop� ND-1000 UV/Vis (Wilmington, DE, USA). A detaileddescription of the procedure can be found in Ref. [56]. 4 mg offunctional Fab was recovered from the column. This means thatabout 95% of the protein recovered from the Talon IMAC columnwere active. It should be noted that the binding capacity of thesupport material was no limiting factor in this experiment as it issignificantly higher than 4 mg of antibody.

ELISA: 96-well Maxisorp polystyrene microtiter plates (Nunc,Rochester, NY) were coated with 100 lL/well of a 1 lg/mL solutionof either the BSA-conjugates of p-amino-D-phenylalanine (p-azo-D-Phe-BSA) or p-amino-L-phenylalanine (p-azo-L-Phe-BSA), orunderivatized BSA in 50 mM carbonate buffer, pH 9.6. After over-night incubation at 4 �C, plates were washed three times withPBS/Tween 20. Unoccupied sites on the plate surface were blockedusing 250 lL per well of PBS/Tween 20 containing 1% gelatin.Plates were then incubated for 2 h at 37 �C, followed by threewashes with PBS/Tween 20.

In noncompetitive tests, serial dilutions of Fab in PBS (100 lL/well) were applied to the plate and incubated for 2 h at 37 �C. Afterwashing three times as described above, goat anti-mouse F(ab0)2

secondary antibody labeled with HRP was added (1/5000 in PBS;100 lL/well). Following incubation for 2 h at 37 �C and anotherwashing step, the substrate o-phenylenediamine (Sigma, St. Louis,MO; citrate/phosphate buffer, pH5, 100 lL/well) was added. Absor-bance at 492 nm was read in a FLUOstar plate reader (BMG Lab-technologies, Offenburg, Germany) following acidification with1 N sulfuric acid (100 lL/well).

In competitive ELISAs, the coating, blocking, and washing stepswere performed as described above. However, primary antibody,i.e., Fab, was applied at a constant concentration (50 lL/well in

PBS) and incubated (2 h at 37 �C) in the presence of competitorsat varying concentrations (50 lL/well). As competitors, the pureenantiomers of a number of free amino acids were used. Absor-bance values were converted into % inhibition using the equation:% inhibition = [1�(A/A0)] � 100, where A represents values in thepresence of competitor, while A0 is the absorbance measured inthe absence of competitor. All tests were performed at least intriplicate.

Results and discussion

In this study, enantioselective Fab fragments were generatedutilizing the mRNA of the monoclonal anti-L-amino acid antibody80.2 (anti-L-AA 80.2) that had been produced previously followingclassical immunological methods [45,46,57].

The DNA sequence of the anti-L-amino acid antibody 80.2 wascloned into the pEXP5-CT/TOPO expression vector, and the ex-pressed protein was characterized using SDS–PAGE, ELISA, westernblot, and dot blot analysis.

Sequence analysis of the parent antibody

In order to amplify the complementary DNA encoding the var-iable regions of the heavy and light chains of anti-L-AA 80.2,degenerate primers targeting the framework region one (FR1)and the beginning of the constant region (C1) were employed ina polymerase chain reaction (PCR) [47]. Following sequence analy-sis and conversion into the corresponding amino acid sequence,the VH and VL sequences shown in Fig. 1 were obtained.

The Basic Local Alignment Search Tool (BLAST) was used tocompare the nucleotide and amino acid sequences of VH and VL

of anti-L-AA 80.2 (80.2 VH and VL) with those contained in the Gen-Bank database [58]. The results from the BLAST search databaseconfirmed that both 80.2 VH and VL are from mouse (Mus muscu-lus); the highest score (Bits) for the 80.2 VH was 466 with an E-va-lue of 7e-129, while the highest score (Bits) for 80.2 VL was 509with an E-value of 5e-142.

Production of stereoselective antibody fragments by molecularbiological techniques

The method for constructing Fab antibodies consists of thefollowing:

(1) Primary PCR amplification of VH, VL, CL and CH1 gene frag-ments from 80.2 hybridoma cDNA; (2) Joining of the VH and CH1,and VL and CL sequences via overlap PCR to create HC and LC frag-ments, respectively; (3) Cloning of the HC and LC gene fragmentsinto separate pEXP5-CT/TOPO expression vectors; (4) Electropora-tion, growth and expression of the recombinant vectors inBL21(DE3) cells.

Based on the sequence analysis of the parent antibody, primerswere designed to amplify the DNA sequences of 80.2 VH and VL byPCR (see section Experimental). Analysis of the PCR products in a0.9% agarose gel showed bands for the VH and VL DNA sequencesat about 385 bp and 350 bp, respectively, which is in agreementwith the expected sizes.

Since reliable mouse constant region primers could not befound in the literature, and CH1 is highly conserved, a number ofpreviously published mouse heavy chain sequences were com-pared. These included oligonucleotides from murine constantregion CH1 primers, [59–61] amino acid and nucleotide sequencesfrom various murine CH1 fragments, [62–64] and the DNA se-quence IGG1 #37 from the Kabat database [53]. A BLAST searchconfirmed that all these sequences originated from murine IgGantibody. The sequence IGG1 #37 is almost identical with the

Fig. 1. Amino acid sequences of the VH and VL domains of anti-L-AA 80.2. CDRs 1–3 are shown in bold.

24 P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29

mouse antibody sequence BC024405.1 of the BLAST database butlacks about 18 nucleotides at the 30 end. Because of the high degreeof homology of the variable domains of 80.2 and BC024405.1 theCH1 nucleotide sequence of the latter was selected as templatefor the design of the primers CH1_fw_constant and CH1_rev_con-stant, which were used for amplifying the CH1 sequence of anti-L-AA 80.2. Agarose gel electrophoresis of the PCR products showedthe heavy constant fragment at about 359 bp, which is in agree-ment with the expected size. The DNA encoding the CH1 genewas confirmed by sequencing, and a BLAST search verified thatthe sequence (Fig. 2a) was consistent with mouse CH1 with a Bitsscore of 579 and an E-value of 6e-163.

A similar strategy was applied to obtain mouse light chainconstant region (CL) primers. Several published sequences werecompared, namely a complete murine kappa chain constant se-quence, oligonucleotides from murine CL primers, [59,60,64] aminoacid and nucleotide sequences from various murine CL fragments,[65,66] and the sequence MOPC 21 # 8 from the Kabat database[53]. Among the query sequences, MOPC 21 # 8 resulted in the bestmatch with the mouse CL sequence AB048524 (RefSeq ID); thehighest score (Bits) was 588 and the E-value was 1e-165. Align-ment between MOPC 21 # 8 and AB048524.1 showed that thesequences are in fact identical, which again is not surprising sincealso CL is highly conserved. The MOPC 21 # 8 sequence wastherefore used to design the primers CL_fw and CL_rev. Using theseprimers and cDNA of anti-L-AA 80.2, the CL region could success-fully be amplified by PCR yielding a DNA product of about321 bp, which was subsequently gel purified and sequenced (seeFig. 2b).

Construction of the heavy chain gene fragment (HC) was carriedout by overlap extension PCR. To allow generation of the whole80.2 HC sequence by PCR, the two fragments, 80.2 VH and 80.2CH1, should ideally overlap by 15–35 bp. For this reason, the fol-lowing forward primer, CHextension80.2VH, was designed whichcontains the antisense of the 80.2 VH 30 end at its 50 end. The

Fig. 2. Nucleotide and amino acid seque

80.2 CH1 DNA was then amplified and the 33 nucleotides of 80.2VH were added to the 50 end of the 80.2 CH1 sequence. As a conse-quence, the 50 end of 80.2 CH1 and the 30 end of 80.2 VH hybridizedduring the overlap PCR to form a complete 80.2 HC monocistronicgene fragment (Fig. 3) with a length of about 670 bp.

The produced 80.2 HC DNA fragment was sequenced in itsentirety at the Core DNA Analysis Lab at Northern Illinois Univer-sity (Fig. 4a).

Similarly to the HC fragment, the 80.2 LC fragment was con-structed by joining VL and CL in an overlap extension PCR. PrimerCL.o/v.80.2VL was designed with a 20 bp overhang complementaryto the 30 end of the 80.2 VL complement region. Upon PCR amplifi-cation, a complete 80.2 LC gene of approximately 645 bp consistingof VL and CL was obtained and sequenced (Fig. 4b) to confirm theopen reading frame.

Making use of the terminal transferase activity of Taq polymer-ase, amplification of 80.2 HC and LC fragment DNA added singledeoxyadenylate (A) residues to the 30 ends of the PCR products.These were utilized in TOPO cloning reactions to form A-T hybridswith the expression vector. In addition, new forward primers weredesigned containing overhang nucleotides, ‘‘CCATG’’, to be incorpo-rated at the 50 ends of both 80.2 HC and LC DNA fragments. Addi-tion of the start codon ‘‘ATG’’ to the overhang was necessary tofacilitate proper translation of the recombinant protein. The ‘‘CC’’was incorporated to ensure proper spacing between the ribosomebinding site and the initial ATG codon for the desired protein.

Since LC fragments have a tendency to form homo-dimers,known as Bence Jones proteins, [67,68] a reverse primer was de-signed to contain a premature stop codon at the 30 end preventingprotein translation beyond the LC fragment. As a consequence, theLC protein would be free of an incorporated His-tag at the C-termi-nus, which is part of the pEXP5-CT/TOPO plasmid. This approachwas intended to prevent co-purification of LC protein or LChomo-dimers along with correctly associated Fab antibody on ametal affinity column.

nces of (a) 80.2 CH1 and (b) 80.2 CL.

80.2 VH CH1e

80.2 VH

CH1e

80.2 HC

A

B

Fig. 3. PCR reactions used for the assembly of 80.2 HC. 80.2 VH and 80.2 CH1 genesegments were first produced separately using cDNA from hybridoma cells. Overlapareas (shaded) complimentary to the end of 80.2 VH were created at the beginningof CH1 segments, resulting in an extended CH1 (CH1e). Overlap extension PCR usingprimers A and B yielded the complete 80.2 HC sequence.

P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29 25

The pEXP5-CT/TOPO recombinant vectors containing the 80.2HC and LC gene fragments, respectively, were separately trans-formed into BL21(DE3) E. coli cells, which facilitates high levelsof protein expression due to the tight control of the T7 promoter.Transformed cells were screened for the presence and correct

Fig. 4. Nucleotide and corresponding amino acid sequences of (a) 80.2 HC from VH and CH

regions in bold.

orientation of the HC and LC genes; this was necessary as TOPOcloning is non-directional due to the T-A hybridization.

Both the 80.2 HC and 80.2 LC antibody proteins were expressedseparately. A positive colony for each fragment was used to inocu-late an overnight culture. Protein expression was induced withIPTG and whole cell lysates were extracted using the B-PER II Bac-terial Protein Extraction Reagent and unfolding/refolding buffer asdescribed in Experimental, section ‘‘Expression of the Fab protein.’’SDS–PAGE gels showed no difference in the amount of proteinobtained upon cell disruption using B-PER II Bacterial ProteinExtraction Reagent or other lysis buffers (results not shown), butsonication lead to a significant decrease in the amount of protein.Therefore, B-PER II Bacterial Protein Extraction Reagent was usedfor protein extraction. The 80.2 HC and LC fragments both showedbands at approximately 25 kDa (Fig. 5).

In order to optimize the induction time for the production of theprotein fragments, expression yields for HC and LC obtained afterdifferent times were compared. It was found that induction after4 h gave the highest yield of IMAC-purified protein for both frag-ments (Table 1). Since the expression vector was void of a signalsequence, protein should have been retained in the cytoplasm.The basal level of protein expression is likely due to a leaky plas-mid, but expression levels could be improved upon induction withIPTG.

Following insertion of the HC and LC sequences into expressionvectors, both the 80.2 HC and 80.2 LC antibody fragments were ex-pressed separately and isolated from whole cells using B-PER andunfolding buffer [69,70]. They were then mixed at an equal molar

1 and (b) 80.2 LC from VL and CL. Variable regions are shown in regular font, constant

NR NR R

1 2 3

25 kDa

50 kDa

32 kDa

Fig. 5. SDS–PAGE analysis of the refolded 80.2 Fab, purified by IMAC. Lane 1, eluateFab; lane 2, eluate Fab; lane 3, Fab under reducing conditions.

0.8

1.0

1.2

1.4

1.6

1.8

nce

(492

nm

)

26 P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29

ratio in refolding buffer. It was assumed that the individual frag-ments would find each other to produce a whole functional 80.2Fab protein fragment. Antibody formation was investigated bydot blot. As seen in Fig. 6, colored dots were only observed with re-folded samples or positive controls, while neither non-refoldedsamples nor the lysis products of non-transformed BL21(DE3) cellsused as negative control caused color formation. This result indi-cated that a properly assembled and folded Fab fragment had beenformed using the protocol previously described for VHH antibod-ies, [71] which are single domain antibodies engineered from hea-vy-chain antibodies found in camelids [71].

The Fab was designed to contain a His-tag at the C-terminus ofthe heavy chain for purification on a metal affinity resin. Followingthe Talon Metal Affinity Resin protocol, protein-containingfractions were collected and further analyzed by SDS–PAGE. Bandsobtained under non-reducing conditions at the expected MW of50–55 kDa indicated that the 80.2 HC and LC fragments had indeedsuccessfully refolded and combined to form an Fab (Fig. 5, lane 1and 2). An additional band at 25 kDa can be attributed to HC whichcoeluted with the Fab, as 80.2 HC was expressed with a His-tag. Asexpected, the Fab dissociated into HC and LC fragments underreducing conditions (Fig. 5, lane 3).

The amount of properly folded, active 80.2 Fab that could berecovered from a 1 L culture was approximately 4 mg as estimatedby affinity chromatography on an immobilized derivative of p-ami-no-L-phenylalanine following a previously published proce-dure.[56] 95% of the protein eluted from the IMAC column wasactive. Considering the amounts of LC and HC obtained after 4 hof induction (Table 1), this corresponds to a yield of about 1%.Western blot analysis, furthermore, showed that a secondaryanti-mouse antibody was able to recognize the expressed proteinfollowing non-reducing gel conditions (not shown).

Evaluation of the binding specificity of produced antibody fragments

Functionality as well as stereoselectivity of the purified 80.2 Fabantibody was first assessed using noncompetitive ELISA. The 80.2

Fig. 6. Dot blot analysis of the 80.2 Fab upon refolding. Dot 1, non-refolded Fab; dot2, blank, no antibody protein; dot 3, BL21 (DE3) non-transformed cells; dot 4, Fabafter refolding; dot 5, blank; dot 6, 80.2 parent antibody as positive control. 2 lgprotein was blotted for all samples.

Fab stereoselectively bound to the BSA conjugate of the haptenp-amino-L-phenylalanine (p-azo-L-Phe-BSA) that had been usedas solid phase coating, but no binding to the corresponding conju-gate of the D-enantiomer or underivatized BSA was observed(Fig. 7). This is in agreement with the binding properties displayedby the parent a-L-amino acid antibody 80.2 [72].

The interaction between antibodies and antigens is governed byamino acid residues in the hypervariable regions within both theVH and VL. In order to examine the importance of each of the twopolypeptide chains on the stereoselectivity and antigen-bindingcapacity of the 80.2 Fab antibody, HC and LC were also expressed,purified, and tested separately. For this purpose, a His-tag wasincorporated into the LC DNA sequence to facilitate purificationof the 80.2 LC protein by IMAC. Gel analysis following expressionand purification showed bands corresponding to purified HC andLC antibody fragments, respectively (not shown). ELISA experi-ments performed with the HC and LC individual chains did notindicate specific binding activity or stereoselectivity of the individ-ual fragments, which implies that only the combination of bothyields a functional protein.

The assembled 80.2 Fab antibody protein was then further char-acterized in competitive ELISAs using the enantiomers of variousamino acids as inhibitors. In this type of experiment, free aminoacids in solution compete with the antigen-coating on the microti-ter plate for the antibody’s binding site. The concentration of anapplied competitor needed to inhibit antibody binding to theimmobilized antigen correlates with the antibody’s affinity for thiscompound. That is, if the affinity is high, a relatively low concentra-tion of the competitor will be sufficient to decrease antibody bind-ing to the solid-phase immobilized antigen. Similarly, if the affinityis weak, a higher concentration of competitor is required.

ELISA results individually obtained with the D- or L-enantiomersof the amino acids phenylalanine, p-amino-phenylalanine, trypto-phan, and cyclohexylalanine are shown in Fig. 8.

The relative binding affinities of the Fab antibody were deducedfrom the IC50 values (Table 2). These values represent the concen-tration of competitor resulting in a 50% inhibition of antibody-binding to immobilized antigen. Since the original antibody,anti-L-amino acid antibody 80.2, had been raised against an immu-nogen displaying a phenylalanine-based azo-structure on itssurface, it was expected that the Fab would bind more stronglyto competitors that closely resemble the hapten. Thus, it is not

-0.2

0.0

0.2

0.4

0.6

10-fold100-fold

Abso

rba

Dilution of Fab 80.2

Fig. 7. Assessment of the stereoselectivity of refolded 80.2 Fab as determined bynon-competitive ELISA. p-Azo-L-Phe-BSA (j), p-azo-D-Phe-BSA (N) and BSA (d),respectively, were used as solid phase coatings.

10-6 10-5 10-4 10-3 10-2 10-1-20

0

20

40

60

80

100

%In

hibi

tion

concentration [M]10-3 10-2 10-1

0

20

40

60

80

100

% in

hibi

tion

concentration [M]

10-3 10-2 10-1-20

0

20

40

60

80

100

% in

hibi

tion

concentration [M]10-3 10-2 10-1 100 101

0

20

40

60

80

100

% in

hibi

tion

concentration [M]

(a) (b)

(c) (d)

Fig. 8. Results obtained in competitive ELISAs performed with 80.2 Fab antibody using the L-enantiomers (h) or D-enantiomers (j) of (a) phenylalanine, (b) p-aminophenylalanine, (c) tryptophan, and (d) cyclohexylalanine as competitors. Error bars represent standard deviations of triplicate determinations.

P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29 27

surprising that the lowest IC50 values were obtained with L-phen-ylalanine and p-amino-L-phenylalanine (Table 2).

Although L-cyclohexylalanine and L-tryptophan are bound moreweakly by the Fab as indicated by their higher IC50 values, it shouldbe noted that also the interaction of the antibody with these com-pounds is highly stereoselective. The obtained results clearly showthat the Fab only binds to amino acids having the correct (i.e., L-)configuration. In addition, L-amino acids containing different sidechains can be bound, albeit with varying affinities. All these obser-vations are in agreement with previous results obtained with theparent antibody 80.2 and similar antibodies [45,46].

Table 2Relative affinities of Fab 80.2 for various L-amino acids as determined by competitiveELISA.

a-amino acid Structure IC50 (mM)

L-Phenylalanine 2.55

p-Amino-L-phenylalanine 5.04

L-Tryptophan 15.5

L-Cyclohexylalanine 50.1

It is noteworthy that the overall low affinity observed for theinteraction between such antibodies and L-amino acids is noimpediment for their use in analytical applications, but may evenbe desirable, e.g., for enantiomer separation under isocratic elutionconditions [20].

Availability of biotechnologically produced stereoselectiveantigen-binding fragments is expected to greatly facilitate theirutilization in analytical techniques and the structural elucidationof the molecular basis of their chiral discrimination properties.

Acknowledgments

This work was supported by the National Institutes of Health (1R15 GM076000-01). We would like to thank Dr. Scott Grayburnfrom the Core DNA Analysis Lab at NIU for sequencing.

References

[1] W.J. Payne, D.L. Marshall, R.K. Shockley, W.J. Martin, Clinical laboratoryapplications of monoclonal antibodies, Clin. Microbiol. Rev. 1 (1988) 313–329.

[2] N.S. Lipman, L.R. Jackson, L.J. Trudel, F. Weis-Garcia, Monoclonal versuspolyclonal antibodies: distinguishing characteristics, applications, andinformation resources, ILAR J. 46 (2005) 258–268.

[3] G.P. Adams, L.M. Weiner, Monoclonal antibody therapy of cancer, Nat.Biotechnol. 23 (2005) 1147–1157.

[4] H. Panjideh, V. Coelho, J. Dernedde, H. Fuchs, U. Keilholz, T. Thiel, P.M. Deckert,Production of bifunctional single-chain antibody-based fusion proteins inPichia pastoris supernatants, Bioprocess Biosyst. Eng. 31 (2008) 559–568.

[5] L. Sanz, P. Kristensen, B. Blanco, S. Facteau, S.J. Russell, G. Winter, L. Álvarez-Vallina, Single-chain antibody-based gene therapy: inhibition of tumor growthby in situ production of phage-derived human antibody fragments blockingfunctionally active sites of cell-associated matrices, Gene Ther. 9 (2002) 1049–1053.

28 P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29

[6] B.S. Green, Monoclonal antibodies as catalysts and templates for organicchemical reactions, Adv. Biotechnol. Processes 11 (1989) 359–393.

[7] S.C. Sinha, C.F. Barbas 3rd, R.A. Lerner, The antibody catalysis route to the totalsynthesis of epothilones, Proc. Natl. Acad. Sci. USA 95 (1998) 14603–14608.

[8] F. Tanaka, Catalytic antibodies as designer proteases and esterases, Chem. Rev.102 (2002) 4885–4906.

[9] C.E. Cook, Enantiomer analysis by competitive binding methods, second ed., in:I.W. Wainer (Ed.), Drug Stereochemistry: Analytical Methods andPharmocolgy, Marcel Dekker, New York, 1993, pp. 35–64.

[10] H. Hofstetter, O. Hofstetter, Antibodies as tailor-made chiral selectors fordetection and separation of stereoisomers, TrAC – Trend Anal. Chem. 24 (2005)869–879.

[11] O. Hofstetter, H. Hofstetter, M. Wilchek, V. Schurig, B.S. Green, Animmunochemical approach for the determination of trace amounts ofenantiomeric impurity, Chem. Commun. 17 (2000) 1581–1582.

[12] T. Kassa, L.P. Undesser, H. Hofstetter, O. Hofstetter, Antibody-based multiplexanalysis of structurally closely related chiral molecules, Analyst 136 (2011)1113–1115.

[13] H. Silvaieh, M.G. Schmid, O. Hofstetter, V. Schurig, G. Gübitz, Development ofenantioselective chemiluminescence flow-injection and sequential-injectionimmunoassays for a-amino acids, J. Biochem. Biophys. Methods 53 (2002) 1–14.

[14] P. Dutta, C.A. Tipple, N.V. Lavrik, P.G. Datskos, H. Hofstetter, O. Hofstetter, M.J.Sepaniak, Enantioselective sensors based on antibody-mediatednanomechanics, Anal. Chem. 75 (2003) 2342–2348.

[15] A. Tsourkas, O. Hofstetter, H. Hofstetter, R. Weissleder, L. Josephson, Magneticrelaxation switch immunosensors detect enantiomeric impurities, Angew.Chem. Int. Ed. Engl. 43 (2004) 2395–2399.

[16] O. Hofstetter, J.K. Hertweck, H. Hofstetter, Detection of enantiomericimpurities in a simple membrane-based optical immunosensor, J. Biochem.Biophys. Methods 63 (2005) 91–99.

[17] S. Zhang, J. Ding, Y. Liu, J. Kong, O. Hofstetter, Development of a highlyenantioselective capacitive immunosensor for the detection of a-amino acids,Anal. Chem. 78 (2006) 7592–7596.

[18] K. Ariga, G.J. Richards, S. Ishihara, H. Izawa, J.P. Hill, Intelligent chiral sensingbased on supramolecular and interfacial concepts, Sensors 10 (2010) 6796–6820.

[19] Y. Fu, Q. Chen, J. Zhou, Q. Han, Y. Wang, Enantioselective recognition ofmandelic acid based on c-globulin modified glassy carbon electrode, Anal.Biochem. 421 (2012) 103–107.

[20] O. Hofstetter, H. Lindstrom, H. Hofstetter, Direct resolution of enantiomers inhigh-performance immunoaffinity chromatography under isocraticconditions, Anal. Chem. 74 (2002) 2119–2125.

[21] E.J. Franco, H. Hofstetter, O. Hofstetter, Enantiomer separation of a-hydroxyacids in high-performance immunoaffinity chromatography, J. Pharm. Biomed.Anal. 46 (2008) 907–913.

[22] O. Hofstetter, H. Lindstrom, H. Hofstetter, The effect of the mobile phase onantibody-based enantiomer separations of amino acids in high-performanceliquid chromatography, J. Chromatogr. A 1024 (2004) 85–95.

[23] J.M. Zeleke, G.B. Smith, H. Hofstetter, O. Hofstetter, Enantiomer separation ofamino acids in immunoaffinity micro LC–MS, Chirality 18 (17) (2005) 544–550.

[24] T.K. Nevanen, L. Söderholm, K. Kukkonen, T. Suorti, T. Teerinen, M. Linder, H.Söderlund, T.T. Teeri, Efficient enantioselective separation of drug enantiomersby immobilised antibody fragments, J. Chromatogr. A 925 (2001) 97–98.

[25] T.K. Nevanen, M.-L. Hellman, N. Munck, G. Wohlfahrt, A. Koivula, H. Söderlund,Model-based mutagenesis to improve the enantioselective fractionationproperties of an antibody, Prot. Eng. 16 (2003) 1089–1197.

[26] C. Ravelet, E. Peyrin, Recent developments in the HPLC enantiomericseparation using chiral selectors identified by a combinatorial strategy, J.Sep. Sci. 29 (2006) 1322–1331.

[27] E.J. Franco, H. Hofstetter, O. Hofstetter, Determination of lactic acidenantiomers in human urine by high-performance immunoaffinity LC–MS, J.Pharm. Biomed. Anal. 49 (2009) 1088–1091.

[28] H. Hofstetter, J.R. Cary, P.P. Eleniste, J.K. Hertweck, H.J. Lindstrom, D.I. Ranieri,G.B. Smith, L.P. Undesser, J.M. Zeleke, T.K. Zeleke, O. Hofstetter, Newdevelopments in the production and use of stereoselective antibodies,Chirality 17 (2005) S9–18.

[29] E. Harlow, D. Lane (Eds.), Antibodies: A laboratory manual, Cold Spring Harbor,NY, 1988 (pp. 628–629).

[30] M.G. Mage, Preparation of Fab fragments from IgGs of different animal species,Methods Enzymol. 70 (1980) 142–150.

[31] R. Huber, Spatial structure of immunoglobulin molecules, Klin. Wochenschr.58 (1980) 1217–1231.

[32] F.W. Putnam, Y.S. Liu, T.L. Low, Primary structure of a human IgA1immunoglobulin IV. Streptococcal IgA1 protease, digestion, Fab and Fcfragments, and the complete amino acid sequence of the alpha 1 heavychain, J. Biol. Chem. 254 (1979) 2865–2874.

[33] J. Klein, V. Horejší, Immunology, Blackwell Publishing, Malden, MA, 1997 (pp.228–229).

[34] E. Keinan (Ed.), Catalytic Antibodies, Wiley-VCH, 2005, pp. 248–249.[35] T. Fujii, T. Ohkuri, R. Onodera, T. Ueda, Stable supply of large amounts of

human Fab from the inclusion bodies in E. coli, J. Biochem. 141 (2007) 699–707.

[36] P. Roben, P.J. Moore, M. Thali, J. Sodroski, C.F. Barbas 3rd, D.R. Burton,Recognition properties of a panel of human recombinant Fab fragments to the

CD4 binding site of gp120 that show differing abilities to neutralize humanimmunodeficiency virus type 1, J. Virol. 68 (1994) 4821–4828.

[37] P.D. Drew, M.T. Moss, M.J. Pasieka, C. Grose, W.J. Harris, A.J. Porter, Multimerichumanized varicella-zoster virus antibody fragments to gH neutralize viruswhile monomeric fragments do not, J. Gen. Virol. 82 (2001) 1959–1963.

[38] M.L. Grossbard (Ed.), Monolclonal Antibody-Based Therapy of Cancer, 15,Marcel Dekker, 1998, pp. 342–355.

[39] P.M. O’Brien, R. Aitken, Methods in molecular biology: antibody phage displaymethods and protocols, Humana Press 178 (2002) 389–395.

[40] R.J. Flanagan, A.L. Jones, Fab antibody fragments: some applications in clinicaltoxicology, Drug Saf. 27 (2004) 1115–1133.

[41] H. Persson, Envenoming by European vipers antivenom treatment – influenceon morbidity, Przegl. Lek. 58 (2001) 223–225.

[42] H. Tachibana, X. Cheng, K. Watanabe, M. Takekoshi, F. Maeda, S. Aotsuka, Y.Kaneda, T. Takeuchi, S. Ihara, Preparation of recombinant human monoclonalantibody Fab fragments specific for Entamoeba histolytica, Clin. Diagn. Lab.Immunol. 6 (1999) 383–387.

[43] T. Ando, T. Yamashiro, Y. Takita-Sonoda, K. Mannen, A. Nishizono, Constructionof human Fab library and isolation of monoclonal Fabs with rabies virus-neutralizing ability, Micr. Immunol. 49 (2005) 311–322.

[44] S.A. Cohen, M.A. Trikha, M.A. Mascelli, Potential future clinical applications forthe GPIIb/IIIa antagonist, abciximab in thrombosis, vascular and oncologicalindications, Pathol. Oncol. Res. 6 (2000) 163–174.

[45] O. Hofstetter, H. Hofstetter, V. Schurig, M. Wilchek, B.S. Green, Antibodies canrecognize the chiral center of free a-amino acids, J. Am. Chem. Soc. 120 (1998)3251–3252.

[46] O. Hofstetter, H. Hofstetter, M. Wilchek, V. Schurig, B.S. Green, Production andapplication of antibodies directed against the chiral center of a-amino acids,Int. J. Bio-Chromatogr. 5 (2000) 165–174.

[47] Z. Wang, M. Raifu, M. Howard, L. Smith, D. Hansen, R. Goldsby, D. Ratner,Universal PCR amplification of mouse immunoglobulin gene variable regions:the design of degenerate primers and an assessment of the effect of DNApolymerase 30–50 exonuclease activity, J. Immunol. Methods 233 (2000) 167–177.

[48] Sequencher, Gene Codes Corporation, Ann Arbor, MI.[49] ExPasy bioinformatics resource portal. Available from: <http://

web.expasy.org/translate/>, accessed 08/12.[50] NCBI BLAST. Available from: <http://www.ncbi.nlm.nih.gov/BLAST/>, accessed

10/12, Standard Nucleotide BLAST [blastn], nr Database.[51] AbCheck-Antibody sequence test. Available from: <http://www.bioinf.org.uk/

abs/seqtest.html>, accessed 10/12.[52] A.C.R. Martin, Accessing the Kabat antibody sequence database by computer,

Proteins: Struct. Funct. Genet. 25 (1996) 130–133.[53] E.A. Kabat, T.T. Wu, M. Reid-Miller, H.M. Perry, K.S. Gottesman, Sequences of

Proteins of Immunological Interest, NIH, 1991 (Publication no. 91–3242).[54] S.W. Fanning, M. Murtaugh, J.R. Horn, A combinatorial approach to engineer a

dual-specific metal switch antibody, Biochemistry 50 (2011) 5093–5095.[55] M. Wilchek, T. Miron, Activation of sepharose with N, N0-disuccinimidyl

carbonate, Appl. Biochem. Biotechnol. 11 (1985) 191–193.[56] T. Kassa, Production, characterization, and applications of stereoselective

antibodies to alpha-hydroxy acids, (Doctoral dissertation), Northern IllinoisUniversity, DeKalb, IL, 2007.

[57] D. Ranieri, D.M. Corgliano, E.J. Franco, H. Hofstetter, O. Hofstetter, Investigationof stereoselectivity of an anti-amino acid antibody using molecular modelingand ligand docking, Chirality 20 (2008) 559–570.

[58] Available from: <http://www.ncbi.nlm.nih.gov/>, accessed 09/12.[59] D.W. Tonge, J. Hennam, A.R. Greene, I.D. Lee, M.D. Edge, Cloning and

characterization of 1116ns19.9 heavy and light chain cDNA and expressionof antibody fragments in Escherichia coli, II. Pharmacokinetics and immuneresponse, J. Natl Cancer Inst. 80 (1993) 937–942.

[60] P.M. O’Brien, R. Aitken, Methods in molecular biology: antibody phage displaymethods and protocols, Humana Press 178 (2002) 39–58.

[61] J. Wang, R.M. Reilly, P. Chen, S. Yang, M.R. Bray, J. Gariépy, C. Chan, J. Sandhu,Fusion of CH1 domain of IgG1 to epidermal growth factor (EGF) prolongs itsretention in the blood but does not increase tumor uptake, Cancer Biother.Radiopharm. 17 (2002) 665–671.

[62] L.J.N. Cooper, A.R. Shikhman, D.D. Glass, D. Kangisser, M.W. Cunningham, N.S.Greenspan, Role of heavy chain constant domains in antibody-antigeninteraction, J. Immunol. 120 (1993) 2231–2242.

[63] Y. Yamawaki-Kataoka, T. Miyata, T. Honjo, The complete nucleotide sequenceof mouse immunoglobin gamma 2a gene and evolution of heavy chain genes:further evidence for intervening sequence-mediated domain transfer, NucleicAcids Res. 9 (1981) 1365–1381.

[64] O. Stoyanov, A. Kister, I. Gelfand, C. Kulikowski, C. Chothia, Geometric invariantcore for the CL and CH1 domains of immunoglobulin molecules, J. Comput. Biol.7 (2000) 673–684.

[65] P.A. Hieter, E.E. Max, J.G. Seidman, J.V. Maizel Jr., P. Leder, Cloned human andmouse kappa immunoglobulin constant and J region genes conserve homologyin functional segments, Cell 22 (1980) 197–207.

[66] W. Altenburger, P.S. Neumaier, M. Steinmetz, H.G. Zachau, DNA sequence ofthe constant gene region of the mouse immunoglobulin kappa chain, NucleicAcids Res. 8 (1981) 971–981.

[67] M. Kirsch, M. Zaman, D. Meier, S. Dübel, M. Hust, Parameters affecting thedisplay of antibodies on phage, J. Immunol. Methods 301 (2005) 173–185.

[68] R.L. Nachman, R.L. Engle, S. Stein, Subtypes of Bence-Jones proteins, J.Immunol. 2 (1965) 295–299.

P.P. Eleniste et al. / Protein Expression and Purification 91 (2013) 20–29 29

[69] T. Fujii, T. Ohkuri, R. Onodera, T. Ueday, Stable supply of large amounts ofhuman Fab from the inclusion bodies in E. coli, J. Biochem. 141 (2007) 699–707.

[70] H. Lilie, Folding of the Fab fragment within the intact antibody, FEBS Lett. 417(1997) 239–242.

[71] Available from: <http://hornlab.niu.edu/labwiki/pmwiki/pmwiki.php/Main/VHH?action=print>.

[72] P.P. Eleniste, Production of stereoselective anti-amino acid antibody fragmentsby molecular biological techniques, (Doctoral Dissertation), Northern IllinoisUniversity, DeKalb, IL, 2008.