The life cycle of hepadnaviruses is characterized by the synthesis of a 3.2 kb partially double-stranded, relaxed-circular DNA (rcDNA) genome by reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). The mechanism for the genomic replication of hepadnaviridae is now understood in a considerable detail (Fig. 1).10, 52
Fig. 1. Hepetitis B virus life cycle.10
V e s i c u l a r t r a n s p o r t
During initiation of infection, the viral rcDNA genome (Fig. 2A) with a HBV DNA polymerase attached to the 5' end of the minus-strand DNA and a short RNA attached to the 5' end of the plus-strand DNA, is converted into covalently closed circular DNA (cccDNA, Fig. 2B). The cccDNA serves as the template for transcription of viral mRNAs in nucleus. Resulting RNAs are translated to give rise to the polymerase, core, pre-surface/surface, and X gene products. One of these RNAs, the pgRNA that is capped, is polyadenylated, and has a large terminal redundancy, serves as the mRNA for the synthesis of core protein (nucleocapsid subunit) and the viral DNA polymerase. In cytoplasm, the viral DNA polymerase binds to the 5' end of its own viral pgRNAs, and the complex is then packaged into nucleocapsids, where viral DNA synthesis occurs. Within this structure, viral DNA synthesis is initiated; following minus-strand DNA synthesis, plus-strand DNA synthesis occurs. Once partially double-stranded DNA has been produced, progeny core bud into intracellular membranes such as the endoplasmic reticulum (ER) or proximal Golgi to acquire their glycoprotein envelops. These nucleocapsids can also migrate to the nucleus to increase the copy number of cccDNA. Enveloped virions are then secreted via the constitutive pathway of vesicular transport.
A. B.
Fig. 2. HBV DNA in viral replication cycle. A. relaxed circular DNA. HBV DNA polymerase is covalently attached minus-strand DNA. DR1 and DR2 indicated. B.
covalently closed circular DNA.52
HBV DNA within the nucleocapsids is synthesized by following mechanism (Fig. 3).
The HBV DNA polymerase binds to the epsilon stem- loop structure near the 5' end of pgRNA to facilitate packaging into nucleocapsids and initiation of reverse transcription by a protein-priming mechanism, utilizing a tyrosine located near the amino terminus of the reverse transcriptase itself (Fig. 3b). Following the synthesis of 4 bases, the polymerase translocates to the 3' end of the RNA template, where the 4 bases can anneal with complementary sequences (Fig. 3c). During the elongation of the minus-strand DNA to the 5' end of the pgRNA, pgRNA is degraded by the RNaseH activity of polymerase (Fig. 3d). When polymerase reaches the 5’ end of the template, its RNaseH activity leaves an RNA oligomer consisting of the 5' end 17 to 18 bases of the pregenome, including the CAP and DR1 (Fig. 3e). The remaining fragment then serves as the primer for plus-strand synthesis, usually following its translocation to DR2, with which it can hybridize because of the sequence identity between DR1 and DR2 (Fig. 3f). When the plus strand reaches the 5' end of the minus strand, a third translocation occurs to circularize the molecule and to allow continued plus-strand DNA elongation (Fig. 3g). This translocation may be facilitated by the short (~9 bases) terminal redundancy on the minus strand. The plus strand is not completed prior to virion release.
b .
d .
e .
f .
g . c . a .
Fig. 3. Viral DNA synthesis in Hepadnavirus DNA replication. See the text for details.10
HBV DNA polymerase as the target for inhibition of replication
HBV DNA polymerase has a central role in the life cycle of HBV and comprises four domains. From the amino terminus, the se are the terminal protein (TP), spacer, reverse transcriptase (RT), and RNaseH domains.10 As HBV DNA polymerase is poorly immunogenic in mice, the production of monoclonal antibodies that recognize this protein is currently limited. Some monoclonal antibodies that have been produced against human HBV DNA polymerase were raised against the TP, spacer and RNaseH domains. Of these antibodies, only TP domain-specific monoclonal antibodies were able to inhibit protein priming reaction.31, 60 These reports suggest that conserved structural features within the TP region of HBV DNA polymerase are important for its proper functioning. Within the TP domain of HBV DNA polymerase, a tyrosine residue is known to be the most important residue in protein priming and initiation of reverse transcription during viral replication.57 However, the previously reported monoclonal antibodies against the TP domain of human HBV polymerase recognize amino acids 8-20 and 20-30 within the TP domain.60 No antibody that recognizes the tyrosine 65 residue of HBV DNA polymerase where the protein-priming reaction occurs, has yet been identified. In order to produce monoclonal antibodies that recognize epitopes around the 65th residue of the TP domain of HBV DNA polymerase, I synthesized the TP-peptide that encompasses amino acid residues 57-80 of the TP domain of HBV DNA polymerase as a target antigen.
Antibody display methods
Antibodies or other proteins that have specific binding activity are useful tools for monitoring protein abundance and activity. During the past decade, several display strategies have been powerful and efficient tools for the selection and evolution of these kinds of proteins.4, 9, 36, 46
In these kinds of techniques, the connection of genotype and phenotype allows the enrichment of specific functional protein by using selection process, e.g. on immobilized target. In such selection systems, the target specific binding protein (phenotype) coupled to the specific sequence information (genotype) of members of libraries will be retained, while non-adherent proteins will be washed away. The gene encoding the selected protein can then be re-amplified for further evolution and analysis. The strategies that have been successfully developed are either cell-dependent, involving, for example, display on the surface of phage,58 bacteria11 or yeast,9, 27 or cell- free, as in the case of ribosome display17, 20 and mRNA display system39, 47 and so on.
The most commonly used technique is phage display in which each bacteriophage displays a unique peptide or protein on its surface to select ligands with high affinities in vitro.1, 24, 42 As would be expected, the affinity of ligands derived from this process increases with increasing complexity (or diversity) of the starting library.44, 55 The starting complexity of phage display lib raries is generally limited to <109 because of the bacterial transformation requirement. This limitation is also applied to other in vivo display systems. To overcome this limitation, various
techniques have been attempted.6, 29, 35, 55
In vitro display
In vitro methods employ well-defined, cell- free biochemical processes at all steps. Potential advantages of these are larger libraries, fewer of the biases caused by cellular expression, more facile application of round-by-round mutagenesis technique.
All of these means of applied evolution have specific advantages and limitations, and can be used in combination to exploit the synergy of the various approaches. The in vitro methods can be categorized as 1) display format: relying on direct physical linkage to associate genotype with phenotype, or 2) compartmentalization format: the co-sequestration of gene and gene product through the selection step. The display format has developed along two lines. The first relies on the continued association of mRNA with ribosomes and nascent protein, and has been called polysome display or ribosome display by different practitioners. This topic will be described later. Other display format was designed to join the mRNA and protein by a covalent bond, and does not require the continued presence of the ribosome to stabilize the complex.
This method, which generally referred to as RNA display or RNA-peptide fusion, was introduced independently by two groups in 1997.39, 47 mRNA display takes advantage of the translation-terminating antibiotic puromycin, which functions by entering the A site of ribosomes and forming a covalent bond with the nascent peptide. By covalently attaching puromycin to the 3’ end of an mRNA, a covalent
link between a polypeptide and its encoding message can be achieved in situ during in vitro translation.32 These mRNA-peptide fusions can then be purified and subjected to in vitro selection. Some in vitro selection systems consisting of DNA-peptide fusion have been developed to stabilize the genotype. There are various type for these DNA-peptide fusions.28, 53 mRNA display procedure requires careful chemical synthesis and critical purification of puromycin-attached oligonucleotides, which must be ligated to the 3’ end of each mRNA in the sequence libraries. Failure to perform these manipulations appropriately leads to a reduction in the diversity of available libraries. Compartmentalized in vitro system was developed by Tawfik and Griffiths14, 54. This system used aqueous droplets in an oil/water emulsion mimicking cellular compartmentalization. This scheme maintains genotype-phenotype linkage and high concentrations of genes, RNA, proteins and substrates for more effective interaction of the components; and provides a much simpler, more defined micro-environment for controlling the selection conditions than does a living cell. Various compartmentalized in vitro systems were introduced7, 13 but these methods still required to be verified for practical use.
Ribosome display
Ribosome display is a one of the in vitro display methods. This method relies on non-covalent ternary complexes of mRNA, ribosome and protein.51 A fusion protein is constructed in which the domain of interest is fused to a C-terminal
tether, such that this domain can fold while the tether is still in the ribosome tunnel.
This fusion construct lacks a stop codon at the mRNA level, thus preventing the release of mRNA and protein from the ribosome. High concentrations of magnesium and low temperature stabilize the ternary complex. In this system, relatively stable protein-ribosome- mRNA complexes, in which individual nascent proteins remain linked to their encoding mRNA, are formed by stalling ribosome at the end of translation. Protein- ribosome- mRNA linkage allows simultaneous selection of a desired protein and its encoding mRNA from a library (Fig. 3). The selected mRNA can then be converted into cDNA by reverse transcription reaction and amplified by polymerase chain reaction (PCR) (Fig. 4).
The size of a ribosome displa y library potentially very large, since the number of ribosome which can be concentrated into the given reaction volume is high. Thus, it should be possible to create very large libraries more quickly than for cell-dependent systems.
Ribosome display was first developed by Mattheakis et al. for the selection of peptides.36 To produce a population of stalled polysomes, agents such as rifampicin or chloramphenicol, which block prokaryotic translation, were used. A pool of DNA sequences encoding 1012 random decapeptides was applied for selection.12 Selection of the antibody fragment was developed by two groups, Hanes and Pluckthun17 and He and Taussig20. Hanes et al. int roduced additional features to prokaryotic system. One was the stalling of the ribosome through the absence of a stop codon. A number of additions were made to improve the yield of mRNA after
the polysome display cycle, including stem loop structures at the 5’ and 3’ ends of the mRNA, vanadyl ribonucleoside complexes as nuclease inhibitors, protein disulfide isomerase for folding of disulfide-bridged proteins, and an antisense nucleotide to inhibit ssrA RNA, which in the prokaryotic system causes release and degradation of proteins synthesized without stop codon. He et al. applied eukaryotic in vitro expression to ribosome display. This methods derives from two experimental results, namely the functional production of single chain antibodies in vitro in rabbit reticulocyte lysates40 and in the absence of a stop codon, individual nascent proteins remain associated with their corresponding mRNA as stable ternary polypeptide-ribosome-mRNA complexes.8, 21 According to the Hanes et al., the rabbit reticulocyte lysate system gave rise to lower amounts of functional complexes, lower enrichment factors.15 However, because the lysate are commercially available and contain a lower intrinsic RNase activity, the use of this system is easier and more convenient than using prokaryotic in vitro expression system. And it is also possible that different proteins might be expressed with different efficiencies in the two translation systems.2, 3, 41
In a model system using two distinct scFv fragments of an antibody, a 109 -fold enrichment of a specific scFv over the nonspecific scFv was achieved by five selection cycles of ribosome display, with an average enrichment of 100 per cycle.17 In a library selection, ribosome display was applied to the selection and simultaneous evolution of a scFv fragment binding with 40 pM affinity to a Gcn3p mutant peptide, using a library prepared from the spleen of immunized mice.16 And Starting from the
human combinatorial antibody library HuCAL, picomolar affinity binders to insulin were selected and evolved during ribosome display selection.18 All selected antibodies had accumulated many mutations during the PCR amplification cycles which is included in the ribosome display protocol, and the affinity of the antibodies had improved up to 40- fold compared to the antibodies initially present in the library.
In a selection against an unusual DNA structure, namely the guanine quadruplex DNA, it was demo nstrated that antibodies with high specificity could be generated by ribosome display.50 Ribosome display selection was applied also to human antibody library using transgenic mice.19 Progesterone-bovine serum albumin was immunized to transgenic mice carrying human immunoglobulin loci in order to develop human antibody response. Human antibody fragment library from the immunized mice was prepared by recombination. The library was expressed in vitro and selected against progesterone-BSA. Selected antibody fragment have the affinity of ~10-8 M. The advantages of ribosome display are that, in comparison to phage display, larger libraries can be constructed without the transformation step, and the libraries can be further diversified by PCR during ribosome display.5 Ribosome display not only have applied for the selection for binding protein to a wide variety of target but also have great potential for the maturation of high-affinity protein binder and of protein stability.25, 26
Fig. 4. The structure of antibody-ribosome-mRNA complex (ARM) complex.
The absence of stop codon prevents release of mRNA and nascent antibody from the ribosome. The constant region of light chain as a spacer region makes the single-chain antibody fold correctly.
ribosome
mRNA
Ck VH
Vk
Single -chain antibody
Translation of mRNA lacking stop codon
Fig. 5. Ribosome display cycle for antibody selection. A. DNA library encoding the antibody fragments. It has genetically fused to a tether, which allows the protein to fold while the tether is still in the ribosomal tunnel. B. in vitro transcription and translation. The resulting construct, which lacks a stop codon, is transcribed in vitro into mRNA and further translated in vitro. A ribosomal pausing is induced during the translation. That result in stable ternary complexes of antibody-ribosome- mRNA (ARM complexes) formed. C. Affinity selection against the antigen on the ELISA plate. The ARM complexes are directly used to select by binding assay on the immobilized target. The mRNA of the bound complexes is rescued by dissociating the ribosome with EDTA. D. RT-PCR. A reverse transcription reaction followed by PCR yields the genetic information of the selected clones. These clones can then be analyzed or used as input for the next selection round.
Purpose and summary of this study
In this study, I established the ribosome display and then selected useful TP-specific scFv by established ribosome display.
Although ribosome display has many advantages theoretically, only 4 reports have been published about antibody selection from a library using ribosome display.16, 18, 19, 50
Moreover, only one of them exploited a eukaryotic translation system.19 To select anti- TP antibody using ribosome display, it is necessary to establish ribosome display and to test that antibody selection by ribosome display using eukaryotic translation system works properly. After establishing ribosome display, anti-DNA antibody, 3D8 scFv that has binding activity to single stranded DNA (ssDNA), anti- TP scFv was selected from immunized mouse library. Synthetic TP-peptide was used to immunize mice for library construction. mRNA library was obtained from the spleen of the immunized mouse. Recombinant antibody library DNA was constructed by RT-PCR and assembly PCR reaction. From this library DNA, the antibody-ribosome-mRNA (ARM) complexes were prepared by in vitro transcription and in vitro translation. ARM complexes were specifically selected against TP-peptide. After enrichment of antibody library to the TP-peptide by four repeated selection, library was inserted to expression vector. The selected scFvs had binding affinity, for not only the TP-peptide, but also for functional HBV DNA polymerase protein expressed from baculovirus-infected insect cells.
The antibody selection by ribosome display using eukaryotic system was
established and this system was successfully applied to select TP-specific antibody from an antibody library.
II. MATERIALS AND METHODS
A. Model system for ribosome display
1. Construction of VH/ê antibody fragments used for the control reaction of ribosome display
As a model system for ribosome display, previously isolated ssDNA binding scFv (3D8) antibody fragment was used.30 In order for the scFv fragments to be fold outside of the putative ribosomal tunnel, the kappa chain constant region was used as a spacer as schematically shown in Fig. 6. For in vitro transcription and translation, the construct contained the T7 promoter and Kozac sequence. For optimal analysis, such as ELISA, polyhistidine (His6) affinity tag was connected to the 5’ end of the construct. The construct was prepared by assembly PCR. The primers for construction were listed in Table 1. First, 3D8 scFv DNA and the constant region were amplified separately. The 3D8 scFv gene was amplified from pIg20 3D8 plasmid by using 10 pmole primer of 5’ HIS3D8/back (5’-GACCACCATGGACCATCATCATCATCATCATGAGGTCCAGCTGCAGCAG-3’) and 3’ 3D8/for (5’-GTTGGTGCAGCATCAGCCCGTTTTATTTCCAGCTTGGTC-3’) in a reaction volume of 50 ㎕ buffer [10 mM Tris-Hcl (pH 8.3), 40 mM potassium chloride, 10 mM DTT, 1.5 mM magnesium chloride] containing 2 U of
pfu DNA polymerase (Bioneer, Daejun, Korea). After 5 min of denaturation at 94 , samples were amplified for 25 cycles (1 min at 94℃, 1 min at 55℃ and 1 min at 72℃). For Ck DNA amplification, RNA was isolated from a mouse spleen. In brief, the spleen from BALB/c mouse was removed and the tissue was teased apart using either sterile forceps and then disrupt between microscope slides glass to produce a single cell suspension of lymphocytes and erythrocytes. Total RNA was extracted from the cell suspension by using RNA extraction kit (Amersham Biosciences, Piscataway, NJ, USA). First strand cDNAs were synthesized from prepared total RNA using superscript II RNaseH- reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Ck DNA was amplified from the cDNA by using 10 pmole primer of 5’
Ck/back (5’-AAACGGGCTGATGCTGCA-3’) and 3’ Ck/for_XmaI (5’-TCCCCCCGGGCTCTAGAACACTCATTCCTGTTGGAGCT-3’) in a reaction volume of 50 ㎕ buffer (10 mM Tris-Hcl (pH 8.3); 40 mM potassium chloride; 10 mM DTT; 1.5 mM magnesium chloride) containing 2.5 U of Taq DNA polymerase (Bioneer Co). After 5 min of denaturation at 94 , samples were amplified for 25 cycles (1 cycle is 30 sec at 94℃, 30 sec at 55℃ and 30 sec at 72℃). 3D8/for and Ck/back primers are designed in such a way that both the scFv DNA at its 3’ end and the spacer DNA at its 5’ end contain an identical sequence of 18 nucleotides. The PCR fragments were purified by QIAexII gel extraction kit (Qiagen, Stanford Valencia, CA, USA) and used for assembly PCR. The assembly PCR was conducted in a 50 ㎕ PCR mixture containing 5U of pfu DNA polymerase for 25 cycles (1 cycle is 1 min at 94℃, 1 min at 55℃ and 1 min at 72℃) with the primer ST7/back and
Ck/for_XmaI. The amplified product was purified by gel extraction and cloning to pUC18. Amplified DNA fragment was cloned into SmaI (New England Biolabs, Beverly, MA, USA) of pUC18 vector. In brief, the amplified DNA fragment was mixed with pUC18 vector in 10 ㎕ of 1x ligation buffer and then incubated at 16 for overnight in the presence of 1 U of T4 DNA ligase (USB, Cleveland, OH, USA).
After the ligations mixture was transformed into E . coli DH5á. Five were picked and grown at 37℃ for overnight in 5 ㎖ of ampicillin containing LB broth. The plasmids
After the ligations mixture was transformed into E . coli DH5á. Five were picked and grown at 37℃ for overnight in 5 ㎖ of ampicillin containing LB broth. The plasmids