Nascent chain, mRNA, and ribosome complexes generated by a pure translation system

Ribosome display is based on the concept that ternary complexes consisting of a nascent chain, ribosome, and mRNA can be generated, thereby establishing the linkage between genotype and phenotype that is essential for evolutionary experiments. With cell extract-based in vitro translation systems, it has been shown that ternary complexes can be generated by omitting the termination codon from the constructs, which can be stabilized at low temperature in the presence of high Mg2+ concentrations. Using an Escherichia coli-based reconstituted in vitro translation system (PURE system), in which all components necessary for the translation reaction were highly purified and reconstituted, ternary complexes could be generated equally well with a variety of sequences at the 3' end of the RNA, even those with a termination codon. Moreover, the generated complexes were stable at temperatures between 4 and 50 degrees C, and are thus highly stable in contrast to previous assumptions.     

PURE ribosome display and its application in antibody technology

Ribosome display utilizes formation of the mRNA–ribosome–polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA–ribosome–polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

单链抗体展示系统研究进展

单链抗体(single chain antibody fragment,scFv)是由抗体重链可变区(variable region of heavy chain,VH)和轻链可变区(variable region of light chain,VL)通过柔性短肽连接组成的小分子,是具有完整抗原结合活性的最小功能片段,包含抗体识别及抗原结合部位。相比于其他抗体,scFv具有分子量小、穿透性强、免疫原性弱、易构建表达等优点。目前,scFv最常用的展示系统主要有噬菌体展示系统、核糖体展示系统、mRNA展示系统、酵母细胞表面展示系统和哺乳动物细胞展示系统等。近年来,随着scFv在医学、生物学、食品安全学等领域的发展,使得其在生物合成和应用研究方面备受关注。本文对近年来scFv展示系统的研究进展作一综述,以期为scFv的筛选及应用提供理论基础。     

Intracellular ribosome display via SecM translation arrest as a selection for antibodies with enhanced cytosolic stability

Ribosome display is a powerful approach for affinity and stability maturation of recombinant antibodies. However, since ribosome display is performed entirely in vitro, there are several limitations to this approach including technical challenges associated with: (i) efficiently expressing and stalling antibodies on ribosomes using cell-free translation mixtures; and (ii) folding of antibodies in buffers where the concentration and composition of factors varies from that found in the intracellular milieu. We have developed a novel method for intracellular ribosome display that takes advantage of the recently discovered Escherichia coli SecM translation arrest mechanism. Specifically, we provide the first evidence that the encoding mRNA of SecM-stalled heterologous proteins remains stably attached to ribosomes, thereby enabling creation of stalled antibody-ribosome-mRNA (ARM) complexes entirely inside of living cells. Since ARM complexes faithfully maintain a genotype-phenotype link between the arrested antibody and its encoding mRNA, we demonstrate that this method is ideally suited for isolating stability-enhanced single-chain variable fragment (scFv) antibodies that are efficiently folded and functional in the bacterial cytoplasm.     

Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity

We generated a single chain Fv fragment of an antibody (scFv) with a binding affinity of about 5 pm to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with error-prone PCR and DNA shuffling and applied off-rate selection by using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range even for peptide targets.     

A pseudoknot improves selection efficiency in ribosome display

The size and diversity of ribosome display libraries depends upon stability of the complex formed between the ribosome, mRNA and translated protein. To investigate if mRNA secondary structure improves stability of the complex, we tested a pseudoknot, originating from the genomic RNA of infectious bronchitis virus (IBV), a member of the positive-stranded coronavirus group. We used the previously-isolated anti-DNA scFv, 3D8, as a target protein. During in vitro translation in rabbit reticulocyte lysate, we observed that incorporation of the pseudoknot into the mRNA resulted in production of a translational intermediate that corresponded to the expected size for ribosomal arrest at the pseudoknot. Complexes containing the mRNA pseudoknot exhibited a higher efficiency of affinity selection than that those without, indicating that the pseudoknot improves stability of the mRNA-ribosome-antibody complex in a eukaryotic translation system. Thus, in order to improve the efficiency of selection, this relatively short pseudoknot sequence could be incorporated into ribosome display.     

Ribosome display: cell-free protein display technology.

Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. It uses the principle of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes), through the formation of stable protein-ribosome-mRNA (PRM) complexes. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA, which is then converted and amplified as DNA for further manipulation, including repeated cycles or protein expression. Ribosome display has a number of advantages over cell-based systems such as phage display; in particular, it can display very large libraries without the restriction of bacterial transformation. It is also suitable for generating toxic, proteolytically sensitive and unstable proteins, and allows the incorporation of modified amino acids at defined positions. In combination with polymerase chain reaction (PCR)-based methods, mutations can be introduced efficiently into the selected DNA pool in subsequent cycles, leading to continuous DNA diversification and protein selection (in vitro protein evolution). Both prokaryotic and eukaryotic ribosome display systems have been developed and each has its own distinctive features. In this paper, ribosome display systems and their application in selection and evolution of proteins are reviewed.     

核糖体展示口蹄疫单链抗体库的构建

目的:构建库容量大、多样性好的核糖体展示口蹄疫单链抗体(scFv)库。方法: 分离口蹄疫病毒免疫的兔脾细胞,提取总RNA,用RT-PCR扩增兔抗体的重链可变区(VH)基因和轻链可变区(VL)基因,同时扩增作为间隔区的兔抗体Ck基因;采用重叠延伸PCR (简称SOE-PCR)技术连接VH-VL基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接pMD18-T载体转化E.coli DH5α大肠杆菌,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达8.21×10¹³的兔源口蹄疫核糖体展示scFv库。结论: 构建的大容量兔源性口蹄疫核糖体展示抗体库可以成为进一步筛选特异性口蹄疫单链抗体的实验平台,为开发诊断性口蹄疫单链抗体奠定了很好的实验基础。     

Selection of scFvs specific for the HepG2 cell line using ribosome display

The aim of this study was to construct a ribosome display library of single chain variable fragments (scFvs) associated with hepatocarcinoma and screen such a library for hepatocarcinoma-binding scFvs. mRNA was isolated from the spleens of mice immunized with hepatocellular carcinoma cell line HepG2. Heavy and k chain genes (VH and k) were amplified separately by RT-PCR, and an anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into the VH/k chain with a specially constructed linker by SOE-PCR. The VH/k chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. In order to isolate specific scFvs, recognizing HepG2 negative selection on a normal hepatocyte line WRL-68 was carried out before three rounds of positive selection on HepG2. After three rounds of panning, cell enzyme-linked immunosorbent assay (ELISA) showed that one of the scFvs had high affinity for the HepG2 cell and lower affinity for the WRL-68 cell. In this study, we successfully constructed a native ribosome display library. Such a library would prove useful for direct intact cell panning using ribosome display technology. The selected scFv had a potential value for hepatocarcinoma treatment.     

Pure translation display

Methods such as monoclonal antibody technology, phage display, and ribosome display provide genetic routes to the selection of proteins and peptides with desired properties. However, extension to polymers of unnatural amino acids is problematic because the translation step is always performed in vivo or in crude extracts in the face of competition from natural amino acids. Here, we address this restriction using a pure translation system in which aminoacyl-tRNA synthetases and other competitors are deliberately omitted. First, we show that such a simplified system can synthesize long polypeptides. Second, we demonstrate "pure translation display" by selecting from an mRNA library only those mRNAs that encode a selectable unnatural amino acid upstream of a peptide spacer sequence long enough to span the ribosome tunnel. Pure translation display should enable the directed evolution of peptide analogs with desirable catalytic or pharmacological properties.     

Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target

Ribosome display is an in vitro selection and evolution technology for proteins and peptides from large libraries. As it is performed entirely in vitro, there are two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations. A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide. The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for analysis. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 extract for in vitro translation, based on the work originally described and further refined in our laboratory. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display is also included in this issue.     

Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

Background

Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes.

Methodology/Principal Findings

In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM₂) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA–ribosome–antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM₂-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM₂ by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.

Conclusions/Significance

The selection of anti-SM₂ specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs.     

Activity-based in vitro selection of T4 DNA ligase

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA.     

Copper chelating anti-inflammatory agents; N1-(2-aminoethyl)-N2-(pyridin-2-ylmethyl)-ethane-1,2-diamine and N-(2-(2-aminoethylamino)ethyl)picolinamide: an in vitro and in vivo study

An in vitro and in vivo study of some copper chelating anti-inflammatory agents for alleviation of inflammation associated with rheumatoid arthritis (RA) has been conducted. Two copper chelating agents, N(1)-(2-aminoethyl)-N(2)-(pyridin-2-ylmethyl)ethane-1,2-diamine ([555-N]) and N-(2-(2-aminoethylamino)ethyl)picolinamide ([H(555)-N]) have been synthesized as their hydrochloride salt; their protonation constants and formation constants with Cu(II), Zn(II) and Ca(II) determined by glass electrode potentiometry at 298K and an ionic strength of 0.15M. Cu(II) formed stable complexes at physiological pH while the in vivo competitors, Zn(II) and Ca(II) formed weak complexes with both chelating agents. Both [555-N] and [H(555)-N] showed better selectivity for Cu(II) than for Zn(II) and Ca(II). Electronic spectra for species formed at physiological pH suggest a square planar geometry. Speciation calculations using a blood plasma model predicted that these copper chelating agents are able to mobilize Cu(II) in vivo, while bio-distribution studies of their (64)Cu(II)-labelled complexes at physiological pH showed tissue accumulation and retention indicating an encouraging biological half life.