聚乙二醇修饰重组人生长激素的初步研究

目的 探讨聚乙二醇(MW20kD)修饰重组人生长激素(rhGH)的反应条件以及修饰产物的纯化方法。方法在不同条件下,将聚乙二醇活性酯与rhGH反应,以单个PEG-GH的比例为指标,用SDS-PACE和薄层扫描方法,确定其在反应产物中所占的比例;采用CM-Sepharose FF离子交换和Sephacry 1S-200分子筛凝胶层析法对修饰产物进行分离纯化。结果聚乙二醇修饰rhGH的反应条件为pH8．0、rhGH与聚乙二醇的比例1：2(mg：mg)、反应时间2．0h;反应产物经两步纯化,所得的单个PEG-GH纯度大于95％。结论 初步确定了聚乙二醇修饰rhGH的反应条件和修饰产物的纯化方法。     

Chemical modification of Vibrio vulnificus metalloprotease with activated polyethylene glycol

Abstract Vibrio vulnificus , an opportunistic human pathogen causing septicemia, produces a metalloprotease which is suspected to be a virulence determinant, but which is labile in vivo due to inactivation by α -macroglobulin. To obtain a derivative which is stable in vivo, the metalloprotease was modified with activated monomethoxy polyethylene glycol. The modified protease retained full activity to a peptide substrate and 10–20% activity to protein substrates, and was resistant to entrapment by α -macroglobulin because of the increased molecular size (approx. 90 kDa). These findings suggest that the modified protease is stable in vivo and may be used to investigate the pathological actions of the protease in the bloodstream.     

Chemical modification of enzymes for enhanced functionality.

The explosion in commercial and synthetic applications of enzymes has stimulated much of the interest in enhancing enzyme functionality and stability. Covalent chemical modification, the original method available for altering protein properties, has now re-emerged as a powerful complementary approach to site-directed mutagenesis and directed evolution for tailoring proteins and enzymes. Glutaraldehyde crosslinking of enzyme crystals and polyethylene glycol (PEG) modification of enzyme surface amino groups are practical methods to enhance biocatalyst stability. Whereas crosslinking of enzyme crystals generates easily recoverable insoluble biocatalysts, PEGylation increases solubility in organic solvents. Chemical modification has been exploited for the incorporation of cofactors onto protein templates and for atom replacement in order to generate new functionality, such as the conversion of a hydrolase into a peroxidase. Despite the breadth of applicability of chemically modified enzymes, a difficulty that has previously impeded their implementation is the lack of chemo- or regio-specificity of chemical modifications, which can yield heterogeneous and irreproducible product mixtures. This challenge has recently been addressed by the introduction of a unique position for modification by a site-directed mutation that can subsequently be chemically modified to introduce an unnatural amino acid sidechain in a highly chemo- and regio-specific manner.     

Chemical modification of proteolytic enzymes by low molecular weight agents

Low-molecular modification of proteolytic enzymes with aldehydes and anhydrides of carboxylic acids as well as with 2,4,6-trinitrobenzene sulphonic acid was studied. Specific activities of the enzymes were found to be dependent on the modification degree of their amino groups. The retaining of high activities in the region of low extents of enzyme modification enabled biocatalysts with activities similar to those of the native enzymes to be prepared.     

Chemical modification of tropomyosin with NBD-chloride

Muscle tropomyosin was modified with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) at several different pH values. NBD-chloride reacts specifically with SH residue at neutral pH but it reacts with both SH residue and amino residues at alkaline pH. The polymerizability of tropomyosin at low ionic strength and the binding property of tropomyosin to F-actin were not affected by the modification of SH residues but they were lost rapidly by the modification of amino groups, in accordance with the previous report [Johnson, P. & Smillie, L.B. (1977) Biochemistry 16, 2264-2269]. By the addition of heavy meromyosin, labeled tropomyosin which could not bind to F-actin recovered the binding ability to F-actin and it could regulate the superprecipitation of actomyosin in the presence of troponin. Further modification of amino groups (labeling ratios more than 5) led to loss of the regulating ability completely.     

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.     

Chemical force microscopy with active enzymes

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.     

Post-production modification of industrial enzymes

Industry has an increasing interest in the use of enzymes as environmentally friendly, highly efficient, and specific bio-catalysts. Enzymes have primarily evolved to function in aqueous environments at ambient temperature and pressure. These conditions however do not always correspond with industrial processes or applications, and only a small portion of all known enzymes are therefore suitable for industrial use. Protein engineering can sometimes be applied to convey more desirable properties to enzymes, such as increased stability, but is limited to the 20 naturally occurring amino acids or homologs thereof. Using post-production modification, which has the potential to combine desirable properties from the enzyme and the conjugated compounds, enzymes can be modified with both natural and synthetic molecules. This offers access to a myriad of possibilities for tuning the properties of enzymes. At this moment, however, the effects of post-production modification cannot yet be reliably predicted. The increasing number of applications will improve this so that the potential of this technology can be fully exploited. This review will focus on post-production modification of enzymes and its use and opportunities in industry.     

New enzymes with potential for PET surface modification

This work describes newly isolated organisms and their potential to modify the surface of polyethylene terephthalate (PET). Out of the different screening processes, four bacterial and five fungal strains were isolated. A PET model substrate was synthesized (bis (benzoyloxyethyl) terephthalate) and used in the screening process, mimicking the polymer in its crucial properties and having the advantage of defined hydrolysis products. On this model substrate, extracellular enzyme preparations from the isolated microorganisms showed a maximum activity of 8.54 nkat/L. All enzyme preparations showed esterase activity on p-nitrophenyl-acetate while no activity was found on p-nitrophenyl decanoate or p-nitrophenyl palmitate. Increased hydrophilicity of PET fabrics after enzyme treatment was found based on rising height and water dissipation measurements.     

New enzymes with potential for PET surface modification

This work describes newly isolated organisms and their potential to modify the surface of polyethylene terephthalate (PET). Out of the different screening processes, four bacterial and five fungal strains were isolated. A PET model substrate was synthesized (bis (benzoyloxyethyl) terephthalate) and used in the screening process, mimicking the polymer in its crucial properties and having the advantage of defined hydrolysis products. On this model substrate, extracellular enzyme preparations from the isolated microorganisms showed a maximum activity of 8.54 nkat/L. All enzyme preparations showed esterase activity on p-nitrophenyl-acetate while no activity was found on p-nitrophenyl decanoate or p-nitrophenyl palmitate. Increased hydrophilicity of PET fabrics after enzyme treatment was found based on rising height and water dissipation measurements.     

Chemical modification of heparin

Heparin was modified at carboxyl groups by reaction with several pharmacologically important amino-containing compounds in aqueous medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. In dependence on the nature of the amine and the ratio of reagents, conjugates containing 36-100% amide and 0-25% isoureidocarbonyl groups were synthesized. Isoureidoarylamide groups are present, along with amide moieties, in the products of heparin modification by hydroxyl-containing aromatic amines. The conjugate of heparin with p-aminobenzoic acid contained oligomeric arylamide.     

Chemical modification of biocatalysts

Although several powerful methods exist for the redesign of enzyme structure and function these are typically limited to the 20 most abundant proteinogenic amino acids. The use of chemical modification overcomes this limitation to allow virtually unlimited alteration of amino acid sidechain structures. If heterogeneous mixtures of enzyme products are to be avoided, however, the required chemistry should be efficient, selective and compatible with aqueous conditions. Recent advances have been made in the modification of proteinases, aminotransferases and redox enzymes.     

Small ubiquitin-like modifier modification of arrestin-3 regulates receptor trafficking

Nonvisual arrestins are regulated by direct post-translational modifications, such as phosphorylation, ubiquitination, and nitrosylation. However, whether arrestins are regulated by other post-translational modifications remains unknown. Here we show that nonvisual arrestins are modified by small ubiquitin-like modifier 1 (SUMO-1) upon activation of β(2)-adrenergic receptor (β(2)AR). Lysine residues 295 and 400 in arrestin-3 fall within canonical SUMO consensus sites, and mutagenic analysis reveals that Lys-400 represents the main SUMOylation site. Depletion of the SUMO E2 modifying enzyme Ubc9 blocks arrestin-3 SUMOylation and attenuates β(2)AR internalization, suggesting that arrestin SUMOylation mediates G protein-coupled receptor endocytosis. Consistent with this, expression of a SUMO-deficient arrestin mutant failed to promote β(2)AR internalization as compared with wild-type arrestin-3. Our data reveal an unprecedented role for SUMOylation in mediating GPCR endocytosis and provide novel mechanistic insight into arrestin function and regulation.     

Chemical modification of heparin

Heparin was modified at carboxyl groups by reaction with several pharmacologically important amino-containing compounds in aqueous medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. In dependence on the nature of the amine and the ratio of reagents, conjugates containing 36–100% amide and 0–25% isoureidocarbonyl groups were synthesized. Isoureidoarylamide groups are present, along with amide moieties, in the products of heparin modification by hydroxyl-containing aromatic amines. The conjugate of heparin with p-aminobenzoic acid contained oligomeric arylamide.     

Chemical modification of neamine

The aminocyclitol antibiotic neamine has been modified by changing the configuration of one or two hydroxyl groups of the aminocyclitol moiety to elucidate the relationship between configuration and antimicrobial activity. 5-Epi-, 6-epi-, and 5,6-diepineamine have been prepared and their antimicrobial activity has been determined against several micro-organisms.