Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization¹ . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used² . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions³ . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies^1,2.Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding⁴ , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A⁵, a small, bispecific molecule with affinity for both HSA and the novel target was identified⁶ .The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination^1,7.     

Evaluation of passive integrated transponder tag retention from two tagging locations in juvenile pallid sturgeon

Population augmentation and propagation of pallid sturgeon (Scaphirhynchus albus) have been a focus of scientists since the mid‐1990s. Various tag types have been used to gain a better understanding of population characteristics, stocking success, and movement patterns. Passive integrated transponder (PIT) tags have been consistently used since the inception of recovery efforts to mark age‐1 and older pallid sturgeon. This tag has been successfully used with other sturgeon species, and tagging protocols for pallid sturgeon were developed in response to those successes. Tag retention rates in pallid sturgeon have been reported as variable or unknown and there has been no formal study to determine PIT tag retention in pallid sturgeon, particularly with age‐1 individuals that are propagated in hatcheries to be stocked into the wild. The objective was to evaluate retention rates of PIT tags inserted into the operculum and along the base of the dorsal fin of age‐1 pallid sturgeon (total fork length [FL] range = 214–358 mm). Tagged sturgeon (n = 80) were held in a large holding tank and inspected for tag loss twice a month for a total of 189 days. Final tag retention was 83% for tags inserted into the operculum and 85% for tags inserted near the dorsal fin. Tags shed from the operculum were from smaller fish (mean FL = 265 mm) and losses occurred during the first 60 days of the experiment. Tags shed from the dorsal site were predominantly from larger individuals (mean FL = 305 mm) and losses were continual throughout the experiment. Future research should determine size‐specific tag retention rates so that hatcheries can maximize retention in either tag placement location.     

Diagnosis of HNF-1α mutations on a PNA zip-code microarray by single base extension

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1α (HNF-1α) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3′ complementarity to the specific mutation site and 5′ complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1α with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.     

Predicting survival outcomes using subsets of significant genes in prognostic marker studies with microarrays

Background  

Genetic markers hold great promise for refining our ability to establish precise prognostic prediction for diseases. The development of comprehensive gene expression microarray technology has allowed the selection of relevant marker genes from a large pool of candidate genes in early-phased, developmental prognostic marker studies. The primary analytical task in such studies is to select a small fraction of relevant genes, typically from a list of significant genes, for further investigation in subsequent studies.     

Suitability of surgically implanted 8-mm passive integrated transponder tags for small-bodied fishes

Innovative tools that benefit conservation are critical as freshwater fishes are lost at unprecedented rates. Mark-recapture methods can characterize population demographics and life-history traits of diverse fishes, but suitable techniques for tagging for individual recognition of small-bodied fishes are rare. Passive integrated transponder (PIT) tag technology may facilitate the tagging of small fishes and early life stages of larger species. However, relatively little research has evaluated the suitability of these small (8.4 × 1.4 mm) tags for many groups of small fishes. Tag loss (retention and survival) and growth of individuals implanted with PIT tags relative to control and sham treatments were compared for eight fishes of differing morphologies. Additionally, the utility of cyanoacrylate to improve tag retention was tested on a subset of taxa. Fish of each species were equally divided and randomly assigned to one of three treatment groups (handled [control], surgical incision [sham], or surgical incision and PIT tag implantation [PIT]). During the 42-d study period, mortalities and expelled tags were counted daily and growth was measured weekly. Researchers can generally expect little tag loss and uncompromised growth rates for a variety of small-bodied fishes; however, initial fish length was related to the magnitude of physiological effects for some taxa (i.e., blacknose dace Rhinichthys atratulus, johnny darter Etheostoma nigrum, juvenile white sucker Catostomus commersonii). Relatively poor survival (<80%) was observed for two benthic species: johnny darter and blacknose dace, often when incision wounds became inflamed before healing. Prevalent tag loss for johnny darter, tadpole madtom Noturus gyrinus, and a dorsal-laterally compressed cyprinid can be reduced by closing the wounds with cyanoacrylate, but with substantially increased mortality rates. This research demonstrates the broad applicability of PIT tagging technology for ecological studies of small-bodied fishes and alleviates many concerns when surgically implanting tags into several fishes.     

Maximum tag to body size ratios for an endangered coho salmon (<Emphasis Type="Italic">O. kisutch</Emphasis>) stock based on physiology and performance

Many coho salmon stocks (Oncorhynchus kisutch) have been in decline during the past three decades. Canada’s most endangered salmon stock, the Thompson River coho salmon, is being studied extensively as managers attempt to reverse these population declines. Investigators are using acoustic telemetry to track the migratory behaviour and survival of the Thompson River (and other) coho salmon stocks. Coho salmon pre-smolts are relatively small compared with salmonid species that are typically studied using acoustic telemetry; therefore the identification of the appropriate sizes of fish and tags to use is critical. This study tested the effects of surgically implanting the three smallest sizes of acoustic tags currently available on the growth, survival, tag retention, swimming performance and physical condition of coho salmon pre-smolts for 300 days post-surgery. Maximum tag size to body size ratios ranged from 15–17% by fork length and 7–8% by mass for the three tag sizes (11 cm fork length for a 6 × 19 mm tag, 12.5 cm for a 7 × 19 mm tag, and 14 cm for a 9 × 21 mm tag). Based on our results, it is unlikely that coho salmon pre-smolts implanted with acoustic transmitters following these size guidelines would have poor survival in studies of freshwater migratory behaviour as a result of the surgery or the tag.     

Subtypes of Madin-Darby canine kidney (MDCK) cells defined by immunocytochemistry: Further evidence for properties of renal collecting duct cells

The Madin-Darby canine kidney (MDCK) cell line has been proposed as a model for studying intercalated (IC) cells of the renal cortical collecting duct. The IC cells are characterized by peanut lectin (PNA) binding capacity, carbonic anhydrase (CA) activity and Cl^-–HCO ₃ ^- exchange mediated by a band 3-related protein. It has been suggested that these properties are also expressed in MDCK cells. So far however, the nature of the specific protein involved in Cl^-–HCO ₃ ^- exchange, the type of CA isozyme and the relationship between these two characteristics and PNA binding, have not been investigated in MDCK cells by immunocytochemical methods. Using two antibodies raised against human erythrocyte band 3 protein and two against human erythrocyte CA I and II isozymes, our study provides evidence that a protein related to band 3 is expressed in about 5% of cultured MDCK cells; these band 3-positive cells do not bind PNA and are not reactive for CAI or CAII. About 30% of the MDCK cells bind PNA, two-thirds of which are also CAII-positive. A majority (about 65%) of MDCK cells is not reactive for the three markers used; their density is increased after incubation with aldosterone. These data indicate (i) that the Cl^-–HCO ₃ ^- exchanger of the MDCK cells could be related to human erythrocyte band 3, (ii) that the CA activity of the MDCK cell line bears antigenic identity with the erythrocyte CA II isozyme and (iii) that the latter is always co-localized with PNA binding. These results provide immunocytochemical evidence for the heterogeneity of the MDCK cell line, which might reflect the cellular heterogeneity encountered in the renal cortical collecting duct. Our data also indicate that clonal selection will be required for future functional studies of the MDCK cells.     

Binding of HIV-1 TAR mRNA to a peptide nucleic acid oligomer and its conjugates with metal-ion-binding multidentate ligands

A peptide nucleic acid (PNA) oligomer and a series of PNA conjugates featuring covalently attached pendant 1,4,7,10-tetraazacyclododecane (cyclen) or bis((pyridin-2-yl)methyl)amine (DPA) moieties have been synthesized that are complementary to regions of the HIV-1 TAR messenger RNA stem-loop. Thermal denaturation studies, in conjunction win with native gel shift assays, suggest that the PNAs “invade” TAR to produce a mixture of two 1:1 PNA–TAR adducts, tentatively assigned as an “open-duplex” structure, in which the TAR stem-loop dissociates and the PNA hybridizes with its RNA complement via Watson–Crick base-pairing, and a triplex-type structure, in which the initially displaced RNA segment is bound to the PNA:RNA duplex through Hoogsteen base-pairing. Thermal denaturation experiments with the TAR sequence and single-stranded RNA and DNA oligonucleotides, both in the presence and in the absence of Zn²⁺ ions, show that the introduction of cyclen or DPA ligand arms into the PNA oligomer leads to a small but reproducible increase in the T _m values. This is attributed to hydrogen-bonding and/or electrostatic interactions between protonated forms of cyclen/DPA and the cognate RNA or DNA oligonucleotide targets. Contrary to expectations, the addition of Zn²⁺ ions did not further enhance duplex formation through binding of Zn(II)–cyclen or Zn(II)–DPA moieties to the complementary RNA or DNA. Native gel shift assays further confirmed the stability increase of the metal-free cyclen- and DPA-modified PNA hybrids as compared with a control PNA sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Intermolecular dynamics studied by paramagnetic tagging

Yeast cytochrome c and bovine adrenodoxin form a dynamic electron transfer complex, which is a pure encounter complex. It is demonstrated that the dynamic nature of the interaction can readily be probed by using a rigid lanthanide tag attached to cytochrome c. The tag, Caged Lanthanide NMR Probe 5, induces pseudocontact shifts and residual dipolar couplings and does not perturb the binding interface. Due to the dynamics in the complex, residual dipolar couplings in adrenodoxin are very small. Simulation shows that cytochrome c needs to sample a large part of the surface of adrenodoxin to explain the small degree of alignment observed for adrenodoxin. The applied method provides a simple and straightforward way to observe dynamics in protein complexes or domain–domain mobility without the need for external alignment media. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells.

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. We describe a modified approach, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kb of homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for abeta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. Our results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.     

Molecular characterisation and genetic mapping of candidate genes for qualitative disease resistance in perennial ryegrass (Lolium perenne L.)

Background  

Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site – leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database.     

Strand-invading linear probe combined with unmodified PNA

Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40 °C at 100 mM NaCl concentration.     

FastTagger: an efficient algorithm for genome-wide tag SNP selection using multi-marker linkage disequilibrium

Background  

Human genome contains millions of common single nucleotide polymorphisms (SNPs) and these SNPs play an important role in understanding the association between genetic variations and human diseases. Many SNPs show correlated genotypes, or linkage disequilibrium (LD), thus it is not necessary to genotype all SNPs for association study. Many algorithms have been developed to find a small subset of SNPs called tag SNPs that are sufficient to infer all the other SNPs. Algorithms based on the r ² LD statistic have gained popularity because r ² is directly related to statistical power to detect disease associations. Most of existing r ² based algorithms use pairwise LD. Recent studies show that multi-marker LD can help further reduce the number of tag SNPs. However, existing tag SNP selection algorithms based on multi-marker LD are both time-consuming and memory-consuming. They cannot work on chromosomes containing more than 100 k SNPs using length-3 tagging rules.     

A model-based approach to selection of tag SNPs

Background  

Single Nucleotide Polymorphisms (SNPs) are the most common type of polymorphisms found in the human genome. Effective genetic association studies require the identification of sets of tag SNPs that capture as much haplotype information as possible. Tag SNP selection is analogous to the problem of data compression in information theory. According to Shannon's framework, the optimal tag set maximizes the entropy of the tag SNPs subject to constraints on the number of SNPs. This approach requires an appropriate probabilistic model. Compared to simple measures of Linkage Disequilibrium (LD), a good model of haplotype sequences can more accurately account for LD structure. It also provides a machinery for the prediction of tagged SNPs and thereby to assess the performances of tag sets through their ability to predict larger SNP sets.     

Expression and purification of recombinant arginine decarboxylase (speA) from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase’s structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His₁₀-ADC (77.3 kDa) was first purified by Ni–NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His₁₀ tag removed recombinant ADC (74.1 kDa)’s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6–9) and temperature (20–80°C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.     

HapBlock: haplotype block partitioning and tag SNP selection software using a set of dynamic programming algorithms

Recent studies have revealed that linkage disequilibrium (LD) patterns vary across the human genome with some regions of high LD interspersed with regions of low LD. Such LD patterns make it possible to select a set of single nucleotide polymorphism (SNPs; tag SNPs) for genome-wide association studies. We have developed a suite of computer programs to analyze the block-like LD patterns and to select the corresponding tag SNPs. Compared to other programs for haplotype block partitioning and tag SNP selection, our program has several notable features. First, the dynamic programming algorithms implemented are guaranteed to find the block partition with minimum number of tag SNPs for the given criteria of blocks and tag SNPs. Second, both haplotype data and genotype data from unrelated individuals and/or from general pedigrees can be analyzed. Third, several existing measures/criteria for haplotype block partitioning and tag SNP selection have been implemented in the program. Finally, the programs provide flexibility to include specific SNPs (e.g. non-synonymous SNPs) as tag SNPs. AVAILABILITY: The HapBlock program and its supplemental documents can be downloaded from the website http://www.cmb.usc.edu/~msms/HapBlock.     

Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations

The detection of rare mutant DNA from a background of wild-type alleles usually requires laborious manipulations, such as restriction enzyme digestion and gel electrophoresis. Here, we describe a protocol for homogeneous detection of rare mutant DNA in a single tube. The protocol uses a peptide nucleic acid (PNA) as both PCR clamp and sensor probe. The PNA probe binds tightly to perfectly matched wild-type DNA template but not to mismatched mutant DNA sequences, which specifically inhibits the PCR amplification of wild-type alleles without interfering with the amplification of mutant DNA. A fluorescein tag (which undergoes fluorescence resonance energy transfer with the adjacent fluorophore of an anchor probe when both are annealed to the template DNA) also allows the PNA probe to generate unambiguous melting curves to detect mutant DNA during real-time fluorescent monitoring. The whole assay takes about only 1 h. This protocol has been used for detecting mutant K-ras DNA and could be applied to the detection of other rare mutant DNAs.     

Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags

Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.     

Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method

Sample solubility is essential for structural studies of proteins by solution NMR. Attachment of a solubility enhancement tag, such as GB1, MBP and thioredoxin, to a target protein has been used for this purpose. However, signal overlap of the tag with the target protein often made the spectral analysis difficult. Here we report a sortase-mediated protein ligation method to eliminate NMR signals arising from the tag by preparing the isotopically labeled target protein attached with the non-labeled GB1 tag at the C-terminus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.