Phage display combinatorial libraries of short peptides: ligand selection for protein purification

A library of heptapeptides displayed on the surface of filamentous phage M13 was evaluated as a potential source of affinity ligands for the purification of Rhizomucor miehei lipase. Two independent selection (biopanning) protocols were employed: the enzyme was either physically adsorbed on polystyrene or chemically immobilized on small magnetic beads. From screening with the polystyrene-adsorbed lipase it was found that there was a rapid enrichment of the library with “doublet” clones i.e. the phage species which carried two consecutive sequences of heptapeptides, whilst no such clones were observed from the screening using lipase attached to magnetic beads. The binding of the best clones to the enzyme was unambiguously confirmed by ELISA. However the synthetic heptapeptide of identical sequence to the best “monomeric” clone did not act as a satisfactory affinity ligand after immobilization on Sepharose. This indicated that the interaction with lipase was due to both the heptapeptide and the presence of a part of the phage coat protein. This conclusion was further verified by immobilizing the whole phage on the surface of magnetic beads and using the resulting conjugate as an affinity adsorbent. The scope of application of this methodology and the possibility of preparing phage-based affinity materials are briefly discussed.     

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.     

Identification of affinity ligands for protein purification from synthetic chemical combinatorial libraries

A method to screen combinatorial libraries for the development of selective ligands for protein affinity chromatographic purification is described. The method is based on the application of parallel combinatorial libraries, and it has several potential advantages. The screening procedure is simple and straightforward, and it does not require the chemical derivatization of the target proteins or even that the target protein be pure. The experiment can also be designed to select binders that are less likely to cause protein denaturation. Feasibility of this approach is demonstrated with a model study of the chromatographic purification of bovine albumin serum (BSA) and Avidin.     

Expression,purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.     

人工锌指核酸酶的设计与表达纯化

设计表达了四个锌指核酸酶,用于切断人基因组中的rRNA基因家族的内部转录间隔序列,造成双链断裂,以此提高针对多位点基因打靶的效率,为后续基因打靶应用于基因治疗研究奠定基础。首先,在人rRNA基因家族ITS1序列中找到两个合适的9 bp长的序列(中间间隔6 bp)为锌指蛋白识别位点,根据识别位点序列每个位点分别设计两个三锌指蛋白。通过设计引物进行重叠延伸PCR得到全长编码锌指蛋白的DNA,分别克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFP,转化大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指融合蛋白的表达与纯化。同时,将限制性内切酶Fok I的切割结构域分别与四个锌指蛋白序列采用PCR拼接后克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFN,转化到大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指核酸酶融合蛋白的表达并纯化。     

Designing repeat proteins: well-expressed,soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins

We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.     

New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries

Peptides that recognize specific cell types promise to be valuable tools both in research and clinical applications. Cell-specific peptides can be useful as drug delivery vehicles, diagnostic agents, affinity reagents for cell purification, gene therapy delivery agents, and research tools to probe the nature of a cell's surface. Recently, cell-specific targeting-peptides have been identified by phage-display selections against purified cell-surface markers, whole cells in tissue culture, and even tissues within live animals. These methods for identifying cell-targeting peptides will certainly increase the tools available to the scientist for cell-specific targeting.     

Identification of four CCCH zinc finger proteins in Xenopus, including a novel vertebrate protein with four zinc fingers and severely restricted expression

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor alpha secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001-1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four Xenopus cDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx8Cx5Cx3H repeats, two of which are YKTEL Cx8Cx5Cx3H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.     

PHD finger protein 8蛋白多克隆抗体的制备、纯化与检测

PHD finger8(PHF8)蛋白是最新发现的一种带有PHD结构域和Jmjc结构域的蛋白。现有研究表明其可能在基因转录、组蛋白去甲基化等方面发挥重要作用。为研究其功能,本研究构建原核表达载体pET41b-PHF8(aa886-936),在大肠杆菌Escherichia coli BL21中诱导表达带有GST标签的PHF8(aa886-936)亲水片段融合蛋白,并纯化该片段作为抗原免疫家兔,再以CNBr活化Sepharose4B微珠纯化抗血清制备PHF8特异性多克隆抗体。Western blotting以及免疫荧光检测表明该抗体具有很好的特异性,同时免疫荧光染色的结果也表明PHF8蛋白定位于细胞核。     

Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium

The human tumour suppressor P53 is a key protein involved in tumour suppression. P53 acts as a "guardian of genome" by regulating many target genes involved in cell cycle regulation, DNA repair and apoptosis. We report the P53 expression by the methylotrophic yeast Pichia pastoris using the methanol inducible AOX1 promoter. We have produced the rP53 in intracellular form as well as secreted using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence in two genetic contexts of Pichia, Mut(s) and Mut(+). The intracellular P53 was successfully produced by Mut(s) (KM71) as well as Mut(+) (X33) strains, however, the secreted form was mainly observed in the Mut(s) strain, despite a higher number of p53 copies integrated in the Mut(+) strain. Interestingly, in Mut(s) phenotype, the medium pH influences markedly the rP53 production since it was higher at pH 7 than 6.     

SAG, a novel zinc RING finger protein that protects cells from apoptosis induced by redox agents

SAG (sensitive to apoptosis gene) was cloned as an inducible gene by 1,10-phenanthroline (OP), a redox-sensitive compound and an apoptosis inducer. SAG encodes a novel zinc RING finger protein that consists of 113 amino acids with a calculated molecular mass of 12.6 kDa. SAG is highly conserved during evolution, with identities of 70% between human and Caenorhabditis elegans sequences and 55% between human and yeast sequences. In human tissues, SAG is ubiquitously expressed at high levels in skeletal muscles, heart, and testis. SAG is localized in both the cytoplasm and the nucleus of cells, and its gene was mapped to chromosome 3q22-24. Bacterially expressed and purified human SAG binds to zinc and copper metal ions and prevents lipid peroxidation induced by copper or a free radical generator. When overexpressed in several human cell lines, SAG protects cells from apoptosis induced by redox agents (the metal chelator OP and zinc or copper metal ions). Mechanistically, SAG appears to inhibit and/or delay metal ion-induced cytochrome c release and caspase activation. Thus, SAG is a cellular protective molecule that appears to act as an antioxidant to inhibit apoptosis induced by metal ions and reactive oxygen species.     

Successful identification of novel agents to control infectious diseases from screening mixture-based peptide combinatorial libraries in complex cell-based bioassays

Mixture-based peptide synthetic combinatorial libraries (SCLs) represent a valuable source for the development of novel agents to control infectious diseases. Indeed, a number of studies have now proven the ability of identifying active peptides from libraries composed of thousands to millions of peptides in cell-based biosystems of varying complexity. Furthermore, progressing knowledge on the importance of endogenous peptides in various immune responses lead to a regain in importance for peptides as potential therapeutic agents. This article is aimed at providing recent studies in our laboratory for the development of antimicrobial or antiviral peptides derived from mixture-based SCLs using cell-based assays, as well as a short review of the importance of such peptides in the control of infectious diseases. Furthermore, the use of positional scanning (PS) SCL-based biometrical analyses for the identification of native optimal epitopes specific to HIV-1 proteins is also presented.