Chemical micropatterning of polycarbonate for site-specific peptide immobilization and biomolecular interactions

Polycarbonate (PC) is a useful substrate for the preparation of microfluidic devices. Recently, its utility in bioanalysis has attracted much attention owing to the possibility of using compact discs as platforms for the high-throughput analysis of biomolecular interactions. In this article we report a novel method for the chemical micropatterning of polycarbonate based on the printing of functionalized silica nanoparticles. The semicarbazide groups present on the surface of the nanoparticles were used for the site-specific semicarbazone ligation of unprotected peptides derivatized by an alpha-oxoaldehyde group. The peptide micropatterns permitted the specific capture of antibodies. We report also the characterization of micropatterns on PC by using a wide-field optical imaging technique called Sarfus; this allows the detection of nm-thick films by using nonreflecting PC substrates and an optical microscope working with reflected differential interference contrast. The method described here is an easy way to modify polycarbonate surfaces for biomolecular interaction studies and should stimulate the use of PC for developing plastic biosensors.     

酶定向固定化方法及应用的研究进展

传统固定化方法常会导致酶活性大幅度下降,回收率较低,而酶定向固定由于固定化后可完全暴露其活性部位,因而可以保持较高的酶活回收率。本文主要综述了定向固定化酶的两种方法,分别为共价定向固定和非共价定向固定。其中非共价定向固定化主要是抗体与抗原、亲和素/链霉亲和素和生物素以及组氨酸标签与Co2+/Ni2+之间的亲和作用;共价定向固定化主要是通过半胱氨酸残基上的巯基与载体作用。简述了其在生物传感器、分子识别、酶生物燃料电池及酶纯化方面的应用。最后指出今后的主要研究方向为探索新的定向固定化标签以降低对酶活性部位的影响,应用新的载体以提高固定化酶酶活回收率,优化固定化过程及简化固定化步骤等。     

Click chemistry for rapid labeling and ligation of RNA

The copper(I)-promoted azide-alkyne cycloaddition reaction (click chemistry) is shown to be compatible with RNA (with free 2'-hydroxyl groups) in spite of the intrinsic lability of RNA. RNA degradation is minimized through stabilization of the Cu(I) in aqueous buffer with acetonitrile as cosolvent and no other ligand; this suggests the general possibility of "ligandless" click chemistry. With the viability of click chemistry validated on synthetic RNA bearing "click"-reactive alkynes, the scope of the reaction is extended to in-vitro-transcribed or, indeed, any RNA, as a click-reactive azide is incorporated enzymatically. Once clickable groups are installed on RNA, they can be rapidly click labeled or conjugated together in click ligations, which may be either templated or nontemplated. In click ligations the resultant unnatural triazole-linked RNA backbone is not detrimental to RNA function, thus suggesting a broad applicability of click chemistry in RNA biological studies.     

Monitoring the cellular surface display of recombinant proteins by cysteine labeling and flow cytometry

A general method is described that allows one to follow the surface display of recombinant proteins in Escherichia coli without having to use specific antibodies or enzymatic reactions. The method is based on cysteine-specific labeling through Michael addition to the double bond of maleimide and its derivatives, and takes advantage of the fact that naturally occurring surface proteins in E. coli contain no accessible cysteine residues. The method is easy to perform and could be simply applied to different analytic procedures including Western blot, spectral photometry, and flow cytometry. By using this new labeling method, single cells bearing a distinct protein at the surface could be selected by fluorescence-activated cell sorting. The data were obtained by using autodisplay, an efficient surface display system established for E. coli, but the method presented here represents rather a general solution for analyzing the surface display of recombinant proteins, independent of the cellular system used.     

Arsenical grafted membranes for immobilization of thioredoxin-like proteins

In this work, we describe the cografting of glycidyl methacrylate and dimethyl acrylamide onto a macroporous polysulfone polymer. Aminophenyl arsenical compounds were covalently attached to the copolymer through epoxy ring add-on reactions followed by a 2-mercaptoethanol activation. Thioredoxin and thioredoxin-fusion proteins were immobilized onto this surface and detected by specific antibody recognition. Preservation of native protein folding was confirmed by the detection of the enzymatic activity of an unstable fusion protein. Immobilized fusion protein onto the modified material maintains the enzymatic activity for a longer time, up to two weeks, against the free protein under the same storage conditions that remains active for 2 days.     

Simultaneous purification and site-specific modification of pyrroline-carboxy-lysine proteins

Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.     

新型5-羧基-磺酸基罗丹明的微波合成及对蛋白质的标记作用

利用微波加热技术,以2-磺基-4-羧基苯甲醛和N,N-二烷基-3-氨基酚为原料,在空气气氛中,合成了3种结构新颖的反应性荧光染料———5-羧基-磺基罗丹明类化合物。通过荧光光谱的测定,对5-羧基-磺酸基罗丹明-链霉亲和素复合物与TAMRA和Texas Red-链霉亲和素复合物的荧光强度进行对比分析。结果发现,5-羧基-磺基罗丹明类化合物与链霉亲和素共价结合后,产生强荧光,比目前常用的荧光标记染料更适宜用于标记蛋白质。     

Chemical tags for labeling proteins inside living cells

To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, we now face the challenge of understanding how macromolecular machines, signaling pathways, and other biological networks operate in the complex environment of the living cell. The fluorescent proteins (FPs) revolutionized our ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biology, they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chemical methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chemical tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chemical tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an organic fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chemical tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, we focus on the development and application of chemical tags for studying protein function within living cells. Thus, in our overview of different chemical tagging strategies and technologies, we emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. We highlight recent applications in which the chemical tags have enabled sophisticated biophysical measurements that would be difficult or even impossible with FPs. Finally, we conclude by looking forward to (i) the development of high-photon-output chemical tags compatible with living cells to enable high-resolution imaging, (ii) the realization of the potential of the chemical tags to significantly reduce tag size, and (iii) the exploitation of the modular chemical tag label to go beyond fluorescent imaging.     

Simple fabrication of functionalized surface with polyethylene glycol microstructure and glycidyl methacrylate moiety for the selective immobilization of proteins and cells

This study reported simple surface modification for the immobilization of biomolecules such as proteins and cells onto desired area at micron-scale level. First, thin film composed of glycidyl methacrylate (GMA) was prepared by UV-photopolymerization. Then, the polyethylene glycol (PEG) microstructures which played a role in the prevention of nonspecific binding of biomolecules were fabricated by using micromolding in capillaries (MIMIC). Thus, we could easily obtain an orthogonal surface having biomolecular attraction and repulsion areas. In addition, we could control of the height of prepared PEG microstructures with spin coating or not. For the investigation of feasibility of biomolecule patterning onto the functionalized surface, FITC-BSA and HEK 293 were examined as representative biomolecule models. A functionalized surface with GMA promotes the strong adhesion of biomolecules, and PEG microstructures located on the background prevent nonspecific binding of biomolecules at micron-scale level. The orthogonal difference in surface functionality showed strong possibility of simple patterning of biomolecules. In addition, the proposed method could easily control the size, shape, and height of patterns. It will be useful platform technology for the construction of a biomolecule array.     

Evaluation of different linker regions for multimerization and coupling chemistry for immobilization of a proteinaceous affinity ligand

Alkaline conditions are generally preferred for sanitizationof chromatography media by cleaning-in-place (CIP) protocolsin industrial biopharmaceutical processes. The use of such rigorousconditions places stringent demands on the stability of ligandsintended for use in affinity chromatography. Here, we describeefforts to meet these requirements for a divalent proteinaceoushuman serum albumin (HSA) binding ligand, denoted ABD*dimer.The ABD*dimer ligand was constructed by genetic head-to-taillinkage of two copies of the ABD* moiety, which is a monovalentand alkali-stabilized variant of one of the serum albumin-bindingmotifs of streptococcal protein G. Dimerization was performedto investigate whether a higher HSA-binding capacity could beobtained by ligand multimerization. We also investigated theinfluence on alkaline stability and HSA-binding capacity ofthree variants (VDANS, VDADS and GGGSG) of the inter-domainlinker. Biosensor binding studies showed that divalent ligandscoupled using non-directed chemistry demonstrate an increasedmolar HSA-binding capacity compared with monovalent ligands.In contrast, equal molar binding capacities were observed forboth types of ligands when using directed ligand coupling chemistryinvolving the introduction and recruitment of a unique C-terminalcysteine residue. Significantly higher molar binding capacitieswere also detected when using the directed coupling chemistry.These results were confirmed in affinity chromatography bindingcapacity experiments, using resins containing thiol-coupledligands. Interestingly, column sanitization studies involvingexposure to 0.1 M NaOH solution (pH 13) showed that of all thetested constructs, including the monovalent ligand, the divalentligand construct containing the VDADS linker sequence was themost stable, retaining 95% of its binding capacity after 7 hof alkaline treatment. Received May 12, 2003; revised October 8, 2003; accepted October 21, 2003     

A perspective of surface chemistry

Some highlights in the development of experimental strategies in the study of surface chemistry and catalysis over the last two decades are discussed. The role of adsorbate activation by surface oxygen in providing impetus and direction to research over the last two decades is considered and emphasis given to recent work at Cardiff in exploring the role that surface transients can play in determining reaction pathways. Evidence for very efficient kinetic routes to products but involving immeasurably small concentrations of surface species raises general issues relating to the more traditional approach to catalysis based on for example a Langmuir- Hinshelwood mechanism.     

Recent Developments and Strategies for Mutually Orthogonal Bioorthogonal Reactions

Over the past decade, several different metal-free bioorthogonal reactions have been developed to enable simultaneous double-click labeling with minimal-to-no competing cross-reactivities; such transformations are termed ‘mutually orthogonal’. More recently, several examples of successful triple ligation strategies have also been described. In this minireview, we discuss selected aspects of the development of orthogonal bioorthogonal reactions over the past decade, including general strategies to drive future innovations to achieve simultaneous, mutually orthogonal click reactions in one pot.     

Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation

The utility of single-chain Fv proteins as therapeutic agentswould be realized if the circulating lives of these minimalantigen-binding polypeptides could be both prolonged and adjustable.We have developed a general strategy for creating tailored monoPEGylatedsingle-chain antibodies. Free cysteine residues were engineeredin an anti-TNF-     

Selective labeling of proteins by using protein farnesyltransferase

The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four-residue sequence appended onto the C terminus of a protein, with an alkyne-containing substrate. By using a bio-orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein. FRET was used to calculate a F?rster (R) distance of 40 A between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry.     

Traceless affinity labeling of endogenous proteins for functional analysis in living cells

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional affinity labeling method and allows for real-time monitoring of protein activity. With the high target specificity and biocompatibility of this technique, we have achieved individual labeling and imaging of endogenously expressed proteins in samples of high biological complexity. We also highlight applications in which our current approach enabled the monitoring of important biological events, such as ligand binding, in living cells. These novel chemical labeling techniques are expected to provide a molecular toolbox for studying a wide variety of proteins and beyond in living cells.