Recombinant expression of monovalent and bivalent anti-TNT-antibodies: Evaluation of different expression systems

Recombinant expression of monovalent and bivalent anti-TNT-antibodies: Evaluation of different expression systems
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     J. Serb. Chem. Soc. 73 (2) 139–145 (2008)   UDC 662.237.3:66–95:579.23:579.67   JSCS–3696 Original scientific paper doi: 10.2298/JSC0802139S 139   Recombinant expression of monovalent and bivalent anti-TNT- -antibodies – evaluation of different expression systems MLADEN SIMONOVI Ć 1 *, SVETLANA ZLATANOVI Ć -MILOŠEVI Ć 2 , MIROSLAV M. VRVI Ć 3#  and BRANISLAV SIMONOVI Ć 1   1  Institute for General and Physical Chemistry, Studentski trg 12–16, 11000 Belgrade, 2  Faculty of Mathematics and Natural Sciences, University of Kragujevac, Radoja Domanovi ć a 12, 34000 Kragujevac and 3  Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia (Received 7 February, revised 2 July 2007)  Abstract  : Monoclonal 11B3 anti-TNT (trinitrotoluene) antibody  was expressed as a monovalent and bivalent form using different prokaryotic and eukaryotic expression systems. Recombinant expression in  Escherichia coli , mammalian cells and the methylotrophic yeast  Pichia pastoris  was performed to obtain di-sulfide-linked and glycosylated antibody forms. The generation of antibody and subsequent evaluation of the expression rates were performed using intracel-lular, excretory and periplasmatic expression techniques. All methods involved striving for native expressed antibody with maintenance of its functionality only.  Keywords : TNT; antibody; recombinant expression; 11B3; scFv. INTRODUCTION For protein production in the laboratory, the most suitable prokaryotic sys-tem is the gram-negative bacterium  Escherichia coli  because of its rapid growth in high cell densities, easy generic and availability of a large number of vector systems. The first demonstrated expression of functional fragments of antibodies was for  E. coli  in prokaryotic periplasma. The secretion in periplasma was forced  by genetic fusion of antibody fragments with a signal sequence of some periplas-matic protein. 1,2  The building of disulfide bridges is ensured through the oxida-tive nature of the periplasmatic compartment. 3  Protein expression without a signal sequence results in it remaining in the cytoplasma. Native isolation with ultrasound is possible if the protein is soluble in the cytoplasma. It has to be considered that cytoplasma is not an oxidative compartment, and hence the actual protein must go through redox active systems to achieve its functionality. * Corresponding author. E-mail:   #  Serbian Chemical Society member.  140  SIMONOVI Ć   et al.   In addition to  E. coli , other organisms can be used in which antibody frag-ments were successfully expressed and partially secreted into the medium, which is an enormous advantage for preparative and commercial expression. For easier laboratory expressions, each organism should be matched with  E. coli regarding its ease of handling and diversity of possibilities. In yeasts, the eukaryotic folding and post-translational modifications are merged through simple cultivation. The methylotrophic species  Pichia pastoris  can metabolize methanol as the only carbon source and also secrete and process recombinant proteins. 4,5  The high yields and functionality of the secreted recom- binant immunoglobulins underline the importance of yeasts for this application. For scFv (single chain fragment variable) molecules, yields of over 100 mg l  –1  were obtained, which exceed the expression in  E. coli  by more than hundred times. 6  Without doubt, the usage of mammalian cells (COS, CHO, HEK, etc. ) is the most suitable approach for expression of recombinant proteins. The problems with post-translational modifications are thereby almost excluded. The conver-sion of antibody fragments into complete immunoglobulins of different isotypes and their expression in mammalian cells have been demonstrated many times with no loss of binding activity. 7 − 10  The use of recombinant antibody has significant advantages compared with conventional antibody and its use has become more popular nowadays due to the fact that no animals are required in the manufacturing procedure of the recom- binant antibodies. In addition, the manufacturing time is relatively short com- pared with the conventional method. Moreover, the quality of the final products is higher that those manufactured by the non-recombinant method. In this work, recombinant antibodies specific for TNT were expressed in mono- and bi-valent format in different expression systems. The aim of the work was, on the one hand, to examine the expression efficiency in prokaryotic and eukaryotic systems and, on the other, to use these antibodies for the detection of TNT and its derivatives. The best expressing system should then be used for the commercial production of the most sensitive antibody format for TNT detection. EXPERIMENTAL Vectors The vectors pcDNA3.1+ and pPICZ α -ABC for eukaryotic expression in mammalian cells and yeast  Pichia pastoris  were commercially purchased from Invitrogen Life Techno-logies (Karlsruhe, Germany). The  E. coli  expression vector pET26b(+) was purchased from  Novagen (Schwalbach, Germany) and the phagemid-vector pHEN2 from G. Winter, Center of Protein Engineering, MRC Cambridge, UK.  Antibody fragments The gene for the scFv-fragment 11B3 of mouse srcin, as well as the genes for the C H 2 and C H 3 regions of human IgG was available.    RECOMBINANT EXPRESSION OF ANTI-TNT-ANTIBODIES 141   Oligonucleotides The employed oligonucleotides were synthesized by Metabion (Martinsried) (Table I). TABLE I. Utilized oligonucleotides  Name Sequence 5`-3` Target region Restriction sites 11B3 Bsi for Gatccgtacgtgtgggatggcccaggtgaag 11B3-scFv  BsiW  I   11B3 Asc back Gatcggcgcgccacctaggacggtcagcttg 11B3-scFv  Asc I   11B3 Sfi for (pPICZ α B) Gatcggcccagccggccttatggcccaggtgaag 11B3-scFv Sfi I   11B3 Nde for Ggaattccatatggcccaggtgaagctg 11B3-scFv  Nde I   11B3 Not back (pET) Attcttatgcggccgcccgttttatttccagctt 11B3-scFv  Not  I  Standard molecular biology techniques PCR, ligation, restriction, DNA dephosphorylation, agarose gel electrophoresis, DNA extraction from agarose gels, classic plasmid preparation, alcohol precipitation, DNA quanti-fication, etc.  were performed according to standard protocols. 11    Production of competent E. coli  cells, transformation and expression Electrocompetent  E. coli  cells, transformation, periplasmatic and intracellular expression were performed according to standard protocols. 11-13   Standard techniques in protein biochemistry Immobilized metal ion affinity chromatography for protein purification using a Ni-NTA (nickel nitrilotriacetate) matrix, PAGE (polyacrylamide gel electrophoresis), protein determi-nation, Western blot, dialysis, antibody purification via  the Fc region using protein A/G- -PLUS-agarose, etc.  were generally performed according to standard protocols. 11    Eukaryotic expression in yeast and mammalia Antibody expression in yeast  P. pastoris  and mammalia was performed according to stan-dard protocols. 11,14  For the expression in  P. pastoris,  the standard vector pPICZIgGscFv-Fc, which already contained an α  -factor as a signal sequence and an expression cassette (C H 2 and C H 3 genes), was available. Using restriction sites Sfi I and  Asc I, any scFv can be cloned into the cassette and a dimer IgG  C1, with constant C H 2 and C H 3 regions, is generated as the result of the expression. For expression of the IgG  C1-dimer in mammalian cells, HEK (humane embryo kidney) cells were mostly used. The available vector was pcDNA3.1/Zeo, which contained a CMV  promoter, a rat signal sequence, a gene for zeocine resistance and an expression cassette with the genes for the IgG domains C H 2 and C H 3. RESULTS Starting from the TNT-specific scFv-fragment of mouse srcin, named 11B3, monovalent and bivalent antibodies were generated. The fragment was produced in different formats, i.e. , it was expressed in a monovalent form as scFv and the scFv were further used for the generation of bivalent antibodies. In order to use the scFv in the bivalent form, the IgG ∆ C1 constructs were made. To analyze the  binding properties of antibodies, the free TNP–Tris (trinitrophenol–tris-(hydroxy-methyl)aminomethane), as well as the TNP–protein conjugates were used.  142  SIMONOVI Ć   et al.    Monovalent antibody 11B3-scFv For the cloning and expression of the monovalent antibody, a vector pet26b(+) with a stronger T7-promoter was chosen, in which the amplified 11B3-scFv gene was inserted between the restriction sites  Nde I and  Not  I. During the insertion, the signal sequence was removed, which meant a predisposition for intracellular ex- pression in  E. coli  strain BL21 DE3. The expression was performed for 3 hours at 25 °C using the lactose analog IPTG (isopropyl-  β  - D -thiogalactopyranoside) as inducer. The protein was purified from the cytosol supernatant with a NiNTA-ma-trix and subsequently analyzed by SDS-PAGE and immunoblot. The band of 28 kDa on the membrane for the Western-blot confirmed the successful expression of the antibody (Fig. 1). Fig. 1. Analysis of purified 11B3-scFv using immunoblot. 15 µl of protein probe (1) was se- parated under reducing conditions on a 12 % SDS-gel together with 10 µl of protein marker (M). The presence of protein was confirmed after transfer onto a PVDF-membrane and detection with murine anti-His-IgG-antibody (1: 2500) and anti-mouse-IgG–AP conjugate (1: 2500). The determination of the protein concentration gave a value of 65 µg/ml. The antibody was further biotinylated for usage in the TNP-assay.  Bivalent construct 11B3-IgG  ∆ C1 The bivalent construct 11B3-IgG ∆ C1 is a synthetic molecule obtained after cloning the murine 11B3-gene in the expression cassette with constant C H 2 and C H 3 regions of the human gamma heavy chain. Expression of 11B3-IgG ∆ C1 was  performed parallel in two expression systems, i.e ., yeast  Pichia pastoris  and the human cell line HEK293.  Expression in human HEK293 cells As the expression cassette, the vector pcDNA3.1 was available, which had a strong viral promoter and genes for the constant regions of the human gamma    RECOMBINANT EXPRESSION OF ANTI-TNT-ANTIBODIES 143   heavy chain. The restriction sites  Bsi I and  Asc I were chosen for the cloning of the 11B3-gene. After ligation and transformation, characterization of the clones was  performed with PCR. The obtained amplificates confirmed the cloning of the11B3-scFv insert in the vector. The recombinant DNA was further employed for the production of stable secreting cell lines. From ca.  300 ml expression supernatant, the antibody was  purified using protein A-agarose and analyzed by Western blot (Fig. 2). The elu-ted amount of 4.5 ml gave a total protein quantity of about 410 µg (90 µg/ml); hence the expression rate for the 300 ml culture was about 1.37 µg/ml. The pu-rified protein was directly used in the assay for confirming its functionality. This immunocomponent proved to be very sensitive in the assay for detection of TNT-de-rivatives. Using 60 µl of antibody solution at a concentration of 225 ng/ml, 25 fmol TNP–Tris was identified. Fig. 2. Immunoblot of purified 11B3-IgG ∆ C1. 10 µl of protein marker (M) and 10 µl of pro-tein sample (1) were separated on a 7.5 % SDS-gel under non-reducing conditions. The  presence of protein was verified after transfer onto a PVDF-membrane and detection with anti-human-IgG–AP conjugate (1:2500).  Expression in yeast After ligation of scFv and transformation, the clones were controlled using PCR. One of the selected clones was chosen for inoculation of the overnight culture with zeocine and subsequently for DNA isolation. The isolated DNA was then linearized and inserted into yeast cells using electroporation. After yeast electroporation with yeast DNA, a three-day-expression was  performed with one of selected clones. A very small quantity of the protein was de-tected on a PVDF (poly(vinylidene difluoride))   membrane   with anti-human-IgG (1:2500) conjugated with AP (alkaline phosphatase) or HRP (horseradish pero-xidase). No protein was visualized on an SDS-gel. Accordingly, expression in HEK-cells was preferred.
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