Construction of a dual chain pseudotetrameric chicken avidin by combining two circularly permuted avidins

Two distinct circularly permuted forms of chicken avidin were designed with the aim of constructing a fusion avidin containing two biotin-binding sites in one polypeptide. The old N and C termini of wild-type avidin were connected to each other via a glycine/serine-rich linker, and the new termini were introduced into two different loops. This enabled the creation of the desired fusion construct using a short linker peptide between the two different circularly permuted subunits. The circularly permuted avidins (circularly permuted avidin 5 --> 4 and circularly permuted avidin 6 --> 5) and their fusion, pseudotetrameric dual chain avidin, were biologically active, i.e. showed biotin binding, and also displayed structural characteristics similar to those of wild-type avidin. Dual chain avidin facilitates the development of dual affinity avidins by allowing adjustment of the ligand-binding properties in half of the binding sites independent of the other half. In addition, the subunit fusion strategy described in this study can be used, where applicable, to modify oligomeric proteins in general.     

Design and construction of highly stable, protease-resistant chimeric avidins

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T(m) = 106.4 degrees C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T(m) = 96.5 degrees C) than native avidin (T(m) = 83.5 degrees C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T(m) = 97.5 degrees C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T(m) = 111.1 degrees C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.     

Targeted degradation of beta-catenin by chimeric F-box fusion proteins

Adenomatous polyposis coli (APC) tumor suppressor protein, together with Axin and glycogen synthase kinase 3beta (GSK-3beta), forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of cytoplasmic beta-catenin by ubiquitin-dependent proteolysis. Degradation of phosphorylated beta-catenin is initiated by interaction through the WD40-repeat of a F-box protein beta-TrCP, a component of SCF ubiquitin ligase complex. Mutations in APC, Axin, and beta-catenin that prevent down-regulation of cytoplasmic beta-catenin are found in various types of cancers. In the search for efficient treatment and prevention of malignancies associated with increased levels of cytoplasmic beta-catenin, we created chimeric F-box fusion proteins by replacing the WD40-repeat of beta-TrCP with the beta-catenin-binding domains of Tcf4 and E-cadherin. Expression of chimeric F-box fusion proteins successfully promotes degradation of beta-catenin independently of GSK-3beta-mediated phosphorylation. More importantly, this degradation does not require intact APC protein (pAPC).     

Factors dictating the pseudocatalytic efficiency of avidins

The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective rates of hydrolysis among the different avidins reflect several molecular parameters, including binding-site architecture, the availability of the ligand to solvent and the conformation of the ligand and consequent susceptibility to efficient nucleophilic attack. In avidin, the interaction of BNP with Lys111 and disorder of the L3,4 loop (and consequent solvent availability) together comprise the major driving force behind the hydrolysis, whereas in AVR4 the status of the ligand (the pseudo-substrate) is a major distinguishing feature. In the latter protein, a unique conformation of the L3,4 loop restrains the pseudo-substrate, thereby exposing the carbonyl carbon atom to nucleophilic attack. In addition, due to its conformation, the pseudo-substrate in the AVR4 complex cannot interact with the conserved lysine analogue (Lys109); instead, this function is superseded by polar interactions with Arg112. The results demonstrate that, in highly similar proteins, different residues can perform the same function and that subtle differences in the active-site architecture of such proteins can result in alternative modes of reaction.     

Costimulation of T-cell proliferation by a chimeric B7-antibody fusion protein

 T cells require at least two signals for activation and clonal expansion. The first signal conferring specificity is initiated by interaction of the T cell receptor with peptide-bearing MHC molecules. The second, costimulatory signal can be provided by cell-surface molecules on antigen-presenting cells such as B7-1 (CD80) and B7-2 (CD86), which interact with CD28 on T cells. To direct the costimulatory B7-2 molecule to the surface of tumor cells we have constructed a chimeric fusion protein, which consists of the extracellular domain of human B7-2 fused to a single-chain antibody domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas. This B7-2₂₂₅-scFv(FRP5) molecule, expressed in the yeast Pichia pastoris and purified from culture supernatants, binds to B7 counter-receptors and to ErbB2. B7-2_225-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal, which results in enhanced proliferation of syngeneic T cells. Accepted: 14 October 1997     

Molecular cloning of three structurally related genes for chicken avidin

The chicken genomic library was screened using the 32P-labelled 3'-end of avidin cDNA as a hybridization probe. A positive clone, lambda gAV12201, containing a 15-16 kb insert, was detected. The EcoRI subclones, pgAV0.4, pgAV1.8, pgAV3.3 and pgAV3.7 from the genomic clone were subjected to hybridization and restriction enzyme mapping analysis. The preliminary results suggest the existence of three structurally related genes for chicken avidin. Whether the natural gene is within the subclones can only be established when sequencing analyses of the subclones have been completed.     

可剪切多拷贝抗菌肽融合表达载体的构建

抗菌肽是生物体防御系统产生的一类对外源病菌具有高效杀灭活性的小分子多肽, 在植物抗病基因工程中具有重要的应用价值。Thanatin是刺肩蝽(Podisus maculiventris)成虫经诱导产生的一种抗菌肽, 由21个氨基酸残基组成, 该抗菌肽对革兰阳性、革兰阴性菌以及真菌都有很强的抗菌活性。为研究该抗菌肽转入油菜对菌核病抗性提高的效果, 采用同尾酶反复酶切连接的方法构建了分别含1~5拷贝的Thanatin串联融合表达载体, 并导入农杆菌用于油菜的遗传转化。研究采用引物重叠法扩增并克隆了抗菌肽基因, 并采用了一种在植物体内可被特异性切割的短肽作为连接肽, 使多拷贝融合表达的抗菌肽在植物体内可自动剪切为有功能活性的单个抗菌肽单元, 以增加抗菌肽表达丰度和抗菌肽的稳定性。研究还采用了大豆几丁质酶的信号肽作为引导肽引导多拷贝融合表达的抗菌肽分泌到细胞间隙, 以增强抗菌肽作用效果。     

Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by in vitro gene fusion

Two different lengths of the gene encoding Escherichia coli heat-stable toxin (STa) were fused to the carboxy end of the gene coding for the E. coli heat-labile toxin A-subunit (LTA). The hybrid genes directed expression of chimeric LTA-STa proteins. Association of these chimeras with native heat-labile toxin B-subunit (LTB) resulted in protein complexes that bound to GM1 ganglioside and thereby could be assayed in a GM1 ELISA. The complexes reacted with monoclonal antibodies against either LTA, LTB or STa indicating that the STa and LT epitopes remained immunologically intact after fusion. Genetically constructed chimeric proteins exhibiting LT and STa antigens on the same molecule may represent a promising approach to development of broadly protective immunoprophylactic agents and/or useful immunodiagnostic reagents for diarrhoeal diseases caused by enterotoxinogenic E. coli.     

Identification of oxidized proteins in rat plasma using avidin chromatography and tandem mass spectrometry

The objective in much of the proteomics literature today is to establish the difference between healthy and disease states at the protein level using blood plasma. A critical component in this endeavor is to establish what is normal. The focus of the work reported here was to do this with oxidized proteins that might relate to oxidative stress and oxidative stress-related diseases. Oxidative stress is known to increase markedly in cancer, diabetes, heart disease, and neurodegenerative diseases. Since proteins are one of the targets of ROS, generated by oxidative stress, oxidized proteins are excellent biomarker candidates for these diseases. But first it is necessary to identify oxidized proteins that occur in the healthy state. Healthy rat plasma was used in this study as a source for the identification of naturally oxidized proteins. Freshly drawn blood was treated with biotin hydrazide to selectively derivatize carbonyl groups in oxidized proteins. Oxidized proteins thus biotinylated were separated from the other plasma proteins using avidin affinity chromatography. Affinity selected proteins were further fractionated on a C(8) RP column and fractions collected. The collected fractions were then tryptic digested and the peptides identified using a combination of LC/MS/MS and database searches. One hundred forty-six proteins were identified using 700 signature peptides from the tryptic digested chromatographic fractions. The most frequently encountered proteins in the samples were keratins. Brain and liver were among the organs contributing the most oxidized proteins to plasma followed by heart and kidney.     

Construction of bioactive chimeric MHC class I tetramer by expression and purification of human-murine chimeric MHC heavy chain and beta(2)m as a fusion protein in Escherichia coli

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.     

Construction of in-frame chimeric plant viral genes by simplified PCR strategies

The use of polymerase chain reaction (PCR)-based mutagenesis to create chimeric genes is presently not cost-effective because of the size and number of primers as well as the number of PCRs required. We have developed two strategies based on inverse PCR that exploit limited homologies between two DNA molecules to create in-frame chimeric plant viral genes. This report also contains a compilation of information useful for determining possible restriction sites at a given common dipeptide coding motif between any genes of interest.     

Characterization of the chromophores of pyoverdins and related siderophores by electrospray tandem mass spectrometry

Characteristic fragment ions of the various chromophores of the pyoverdin siderophore family obtained by collision activated dissociation of the [M+2H]²⁺ ions are reported allowing unambiguous identification. Tandem mass spectrometrical studies revealed the existence of the first example of a ferribactin with a succinamide side chain, and they add some information to the problem in which way a malic acid side chain is attached to the chromophore.     

Construction and characterization of a chimeric myoglobin

In order to investigate the functional and structural role of modular structure in globins, we have engineered a chimeric myoglobin (ChimMb) in which the first and third exon come from the gene coding for the sperm whale Mb and the second exon from the gene coding for Aplysia limacina Mb. This ChimMb, fused to the Maltose Binding Protein (MBP) and expressed in Escherichia coli as an apoprotein, binds protoheme in a 1:1 stoichiometric ratio. Based on some functional and spectroscopic properties, we conclude that the central core of the ChimMb (which derives from A. limacina) is native-like. On the other hand, the ChimMb deprived (by proteolytic digestion) of the fused MBP displays a considerably reduced stability. These results suggest that the sperm whale A-G-H nucleus does not contribute significantly to the overall stability of the ChimMb.     

Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1

Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.     

Avidin related protein 2 shows unique structural and functional features among the avidin protein family

Background  

The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.     

Construction of a chimeric aminopeptidase by a combination of gene shuffling and mutagenesis

Three chimeric genes were constructed by gene shuffling of aminopeptidases from Aeromonas caviae and Vibrio proteolyticus. Although expressed chimeric enzymes formed inclusion bodies in Escherichia coli, the introduction of two amino acid mutations into the chimeric genes by site-saturated mutagenesis and a random mutation on error-prone PCR resulted in solubilization of the chimeric enzyme. In addition, active chimeric enzyme showed a different thermostability and thermoactivity to parental enzymes.