A non‐invasive dolphin telemetry tag: Computer design and numerical flow simulation

The impact of devices attached to animals remains a challenge in telemetry studies of dolphins. It was hypothesized that the hydrodynamic design of a tag could provide stable attachment to the dorsal fin by means of resultant hydrodynamic force appearing when a dolphin is swimming. To verify this hypothesis the computer fluid dynamics (CFD) study of tag performance was carried out. A virtual model presenting authentic geometry of a dolphin with tag attached to the dorsal fin was constructed. The same model without tag was used as a reference object to calculate tag impact as regards drag, lift, and moments coefficients. Flow around the models was simulated for the range of velocities as well as the ranges of pitch and yaw angles. It was shown that in 33 of 35 CFD scenarios the streamlined shape of a tag generates the lift force that facilitates keeping a tag attached to the fin. Throughout the set of calculations the tag‐associated drag coefficient does not exceed 4%, which indicates low impact. Data obtained present a baseline for the further development of non‐invasive dolphin telemetry tags.     

Tag loss is a minor limiting factor in sea turtle tagging programs relying on distant tag returns: the case of Mediterranean loggerhead sea turtles

As in many other species, tagging has been routinely conducted for decades in over a hundred sea turtle capture-mark-recapture (CMR) programs worldwide. Tag loss is a key limiting factor because it violates the main assumption in CMR models; however, very few estimates of tag loss exist, and we provide here a review. No published estimations of tag loss are available for the Mediterranean, in spite of intensive tagging since the 1980s. This study aims to provide an estimation of tag loss in loggerhead turtles tagged in the Mediterranean. We modeled 64 tag returns out of ca. 2200 loggerhead turtles tagged at Mediterranean foraging grounds, with mark-recapture intervals up to 11.5 years, in order to estimate a daily tag loss probability of the most used tag applied to the most common turtle species in the region. Five models were evaluated through maximum likelihood estimation. The model with the best fit described a tag loss initially high and then decreasing to a lower asymptote, which is probably due to some defective tag applications. The resulting tag loss (0.15 in the first year and 0.31 after 5 years) was comparable or even lower than those from other areas and/or species and predictions indicate that double tagging can make a turtle identifiable for a long period. Hence, in our tagging program and probably in similar ones as well, tag loss appears to be the less important of the factors affecting tag returns, and efforts in other directions are more likely to improve CMR results.     

RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification

Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide “tag” and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel “RIEDL tag system,” which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-₁₆₉RIEDL₁₇₀). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-₁₆₉RIEDL₁₇₀ were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.     

FMRFamide tagging--how an ionotropic receptor can be used to measure peptide secretion

In this chapter, we describe a technique, FMRFamide tagging, that in principle can be used to measure the release of any sequenced neuropeptide. The method relies upon the addition of an "electrophysiologically active" tag to the prohormone that encodes the neuropeptide of interest. Secretion of the electrophysiological tag (and thus the peptide of interest) is detected by activation of the ionotropic "tag receptor." Both the tagged prohormone and the tag receptor are expressed in the cell type under investigation. Since the tag and the neuropeptide of interest are on the same prohormone they are co-secreted and thus secretion of the tag reflects the co-secretion of the neuropeptide of interest. This method can be used to detect neuropeptide secretion on a millisecond timescale.     

Adjusting survival estimates for premature transmitter failure: a case study from the Sacramento-San Joaquin Delta

In telemetry studies, premature tag failure causes negative bias in fish survival estimates because tag failure is interpreted as fish mortality. We used mark-recapture modeling to adjust estimates of fish survival for a previous study where premature tag failure was documented. High rates of tag failure occurred during the Vernalis Adaptive Management Plan’s (VAMP) 2008 study to estimate survival of fall-run Chinook salmon (Oncorhynchus tshawytscha) during migration through the San Joaquin River and Sacramento-San Joaquin Delta, California. Due to a high rate of tag failure, the observed travel time distribution was likely negatively biased, resulting in an underestimate of tag survival probability in this study. Consequently, the bias-adjustment method resulted in only a small increase in estimated fish survival when the observed travel time distribution was used to estimate the probability of tag survival. Since the bias-adjustment failed to remove bias, we used historical travel time data and conducted a sensitivity analysis to examine how fish survival might have varied across a range of tag survival probabilities. Our analysis suggested that fish survival estimates were low (95% confidence bounds range from 0.052 to 0.227) over a wide range of plausible tag survival probabilities (0.48–1.00), and this finding is consistent with other studies in this system. When tags fail at a high rate, available methods to adjust for the bias may perform poorly. Our example highlights the importance of evaluating the tag life assumption during survival studies, and presents a simple framework for evaluating adjusted survival estimates when auxiliary travel time data are available.     

Polyarginine as a multifunctional fusion tag

Fusion to cationic peptides, such as nonaarginine (R(9)), provides a means to deliver molecular cargo into mammalian cells. Here, we provide a thorough analysis of the effect of an R(9) tag on the attributes of a model protein: bovine pancreatic ribonuclease (RNase A). The R(9) tag diminishes the conformational stability of RNase A (DeltaT(m)=-8 degrees C in phosphate-buffered saline). This effect is nearly mitigated by the addition of salt. The tag does not compromise the enzymatic activity of RNase A. An R(9) tag facilitates the purification of RNase A by cation-exchange chromatography and enables the adsorption of RNase A on glass slides and silica resin with the retention of enzymatic activity. The tag can be removed precisely and completely by treatment with carboxypeptidase B. Finally, the R(9) tag increases both the cellular uptake of RNase A and the cytotoxicity of G88R RNase A, a variant that evades the cytosolic ribonuclease inhibitor protein. Thus, we conclude that polyarginine is a versatile protein fusion tag.     

FMRFamide tagging—how an ionotropic receptor can be used to measure peptide secretion

In this chapter, we describe a technique, FMRFamide tagging, that in principle can be used to measure the release of any sequenced neuropeptide. The method relies upon the addition of an “electrophysiologically active” tag to the prohormone that encodes the neuropeptide of interest. Secretion of the electrophysiological tag (and thus the peptide of interest) is detected by activation of the ionotropic “tag receptor.” Both the tagged prohormone and the tag receptor are expressed in the cell type under investigation. Since the tag and the neuropeptide of interest are on the same prohormone they are co-secreted and thus secretion of the tag reflects the co-secretion of the neuropeptide of interest. This method can be used to detect neuropeptide secretion on a millisecond timescale.     

Minimal FLAG sequence useful in the functional epitope tagging of H-Ras

Epitope tagging can interfere with normal protein function, indicating the need for an unobtrusive epitope tag. The FLAG epitope (DYKDDDDK) was examined for a minimal epitope useful in the tagging of H-Ras. The heptapeptide tag, F7 (MDYKDDD), was found to retain reactivity with M2 and M5 monoclonal antibodies in immunoprecipitation, Western blotting, and immunofluorescence microscopy. The F7 tag did not interfere with Ras stability, EGF stimulation of Ras activation, and downstream phosphorylation of MAPK Erk1/2. Unlike the full FLAG sequence, the F7 tag had minimal effect on the growth properties of H-Ras in a colony-forming assay. The F7 tag may be useful when minimizing the effect of tagging on protein function is an important criterion in the selection of an N-terminal epitope tag.     

Tag jumps illuminated – reducing sequence‐to‐sample misidentifications in metabarcoding studies

Metabarcoding of environmental samples on second‐generation sequencing platforms has rapidly become a valuable tool for ecological studies. A fundamental assumption of this approach is the reliance on being able to track tagged amplicons back to the samples from which they originated. In this study, we address the problem of sequences in metabarcoding sequencing outputs with false combinations of used tags (tag jumps). Unless these sequences can be identified and excluded from downstream analyses, tag jumps creating sequences with false, but already used tag combinations, can cause incorrect assignment of sequences to samples and artificially inflate diversity. In this study, we document and investigate tag jumping in metabarcoding studies on Illumina sequencing platforms by amplifying mixed‐template extracts obtained from bat droppings and leech gut contents with tagged generic arthropod and mammal primers, respectively. We found that an average of 2.6% and 2.1% of sequences had tag combinations, which could be explained by tag jumping in the leech and bat diet study, respectively. We suggest that tag jumping can happen during blunt‐ending of pools of tagged amplicons during library build and as a consequence of chimera formation during bulk amplification of tagged amplicons during library index PCR. We argue that tag jumping and contamination between libraries represents a considerable challenge for Illumina‐based metabarcoding studies, and suggest measures to avoid false assignment of tag jumping‐derived sequences to samples.     

Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.     

Construction of a Chimeric Thermostable Pyrophosphatase To Facilitate Its Purification and Immobilization by Using the Choline-Binding Tag

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.     

Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect

Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tips on improving the efficiency of electrotransfer of target proteins from Phos‐tag SDS‐PAGE gel

The sensitivity of Western blotting analysis after Phos‐tag SDS‐PAGE is occasionally inferior to that after normal (Phos‐tag‐free) SDS‐PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos‐tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet‐tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos‐tag SDS‐PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos‐tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet‐tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos‐tag gels were similar to those from normal gels. In some cases involving the use of a Phos‐tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.     

Increased solubility of integrin betaA domain using maltose-binding protein as a fusion tag

In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15b (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin betaA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the betaA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein.     

Rapid genotyping of three polymorphisms in the LBP gene using a triplex pyrosequencing approach

We described a triplex polymerase chain reaction (PCR) and triplex pyrosequencing assay which allowed a simultaneous determination of three tag single nuleotide polymorphisms (tag SNPs) in the lipopolysaccharide-binding protein (LBP) gene: rs1780623, rs11536972 and rs2232618. This method enables a fast and cost-effective genotyping and a simultaneous determination of the three tag SNPs.     

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background

Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.

Methodology/Principal Findings

Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.

Conclusions and Significance

The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.     

Retention of loop,monel, and passive integrated transponder tags by wild,free‐ranging Lake Sturgeon (Acipenser fulvescens Rafinesque, 1817)

Tagging or marking of fish is instrumental to fisheries biologists and managers seeking to distinguish groups of fish, track movement or migration patterns, and monitor population characteristics. However, tag loss can inhibit the ability of biologists and managers to reach these objectives. The ability of Lake Sturgeon to live for long periods of time and reach large sizes, in combination with their dynamic spawning activity, requires tags to be retained under a variety of environmental and physically demanding conditions. This study evaluated tag retention of loop, monel, and passive integrated transponder (PIT) tags on wild, free‐ranging Lake Sturgeon in Lake Huron and the St. Clair – Detroit River System. Lake Sturgeon in this study were double‐tagged with both a PIT tag under one of the three anterior‐most dorsal scutes and an external tag (loop or monel) at the base of the dorsal fin. Fish were at large for up to 16 years. Overall, tag loss for PIT tags was 1% followed by monel tags at 12% and loop tags at 36%. Tag loss for loop tags was higher when the initial length of Lake Sturgeon tagged was smaller. Tag loss for monel tags increased with time at large but was not related to length at initial tagging. Monel tags left behind abrasion marks when attached to smaller Lake Sturgeon. PIT tag retention was higher than reported in previous studies that tagged other sturgeon species in different body locations. Monel tag retention was higher than other external tag types evaluated in previous studies while loop tags had similar retention rates to external tag types. Most previous studies on tag retention of sturgeon species were of shorter duration and conducted in laboratory settings, therefore loop tags may have performed more favorably during studies under short term laboratory settings. Results of this study suggest that PIT tags inserted below dorsal scutes represent a viable option for long‐term tracking of Lake Sturgeon. Monel tags attached at the base of the dorsal fin also seem to be a viable option relative to other external tag types, but should be limited to larger sturgeon as they can leave behind abrasion marks.     

Long-term retention of dummy acoustic transmitters in adult brown trout

A group of 36 1+ age class Salmo trutta were surgically implanted with dummy acoustic tags and monitored for 370 days. In total 13 tags were expelled throughout the experiment with an overall tag loss rate of c. 0^.035 tags per day. Fish length was the only explanatory variable which had a significant association with subsequent tag expulsion. The estimated probability of retaining a tag for a year for a fish of length 32 cm was 0.76, 34 cm was 0.60 and 36 cm was 0.38. The long-term tag loss patterns were examined and discussed.