Wool Protein Research Articles

Vale Neil McKern - Australian Insulin Receptor Scientist.

Neil Moreton McKern, the first author on the landmark paper describing the crystal structure of the human insulin receptor ectodomain (1), passed away on the 20 March 2014 after a year-long battle with multiple myeloma. Neil McKern was born on 17 December 1946 at Richmond, New South Wales. His career in science began at the age of 18 as a technical assistant to Dr. Fred Stewart in the CSIRO Division of Protein Chemistry in Parkville, VIC, Australia, where he worked on the chemical synthesis of the peptide hormone oxytocin. Neil then studied part time, obtaining a B.Sc. degree in 1971 and a Ph.D. degree in 1981, both from the University of Melbourne. His thesis title was “Serum somatomedin and the regulation of growth-studies with rodent models.” Over the subsequent 10 years at CSIRO, Neil contributed to several aspects of protein science, including the purification of wool proteins for structure determination, the sequencing of the pilin protein of Dichelobacter nodosus (which causes footrot in sheep), the study of the proteins of infectious bursal disease virus (a major poultry pathogen) coupled with development of a recombinant vaccine, and the study of variation in the coat protein sequence across the Potyviridae (the largest family of plant viruses). These projects reflected the agricultural priorities of CSIRO’s science at the time. Figure 1 Neil McKern in 2006. These endeavors laid the foundation for Neil’s involvement starting in 1992 with what was to become a “grand challenge” at CSIRO, namely, Colin Ward’s program to understand the structural biology of the insulin receptor, the Type 1 insulin-like growth factor, and the epidermal growth factor receptor. Spurred on by the successful determination of the structure of the human growth hormone bound to its receptor by Tony Kosiakoff and colleagues at Genentech, the allure of a structure of insulin bound to its receptor was irresistible. However, the complexity of these receptor ectodomains proved to be substantial – high levels of glycosylation, high numbers of disulfide bonds, and the existence of multiple liganded states. Neil masterminded the practicalities of purifying bulk quantities of the ectodomains and truncated ectodomain fragments of these receptors from large (100 l plus) scale mammalian cell culture supernatants. His demands were high – to achieve tens of milligram quantities of receptor protein displaying no more than one band on an isoelectric focusing gel. He set up the first forays of the lab into robotic protein crystallization and the combination of this technology with isoelectrically pure protein finally delivered the first – and still only – structures of the Type 1 IGF-1 receptor L1-CR-L2 fragment (2), the insulin receptor L1-CR-L2 fragment (3), and the intact insulin receptor ectodomain (1). Such care and ingenuity absorbed Neil until 2007, at which point CSIRO terminated the project upon Colin’s retirement. His endeavors also led to structures of the truncated ectodomains of the epidermal growth factor receptor in complex with TGFα (4) and of its co-family member ErbB2 (5). Neil retired from CSIRO in 2010, but not before playing a key role in ensuring that the structural studies of the insulin receptor continued at the neighboring Walter and Eliza Hall Institute of Medical Research in Melbourne. Always striking to all Neil’s colleagues was his cheerful optimism, which was coupled with healthy cynicism about the way in which his employing organization functioned. Part of this optimism derived no doubt from his passion for running, which seemed to not only energize his body but also to provide a wealth of ideas about how to prepare the next sample for crystallization. Giving up on the “grand challenge” would have to Neil been akin to pulling out of a marathon half way along the course. Neil was dedicated to his family, which together faced the challenge of re-building their beloved family retreat in Marysville after it burnt to the ground in the devastating Black Saturday bushfires of 2009. For us, and for the many others who worked with him, his enthusiasm for science, and his collegial support is greatly missed.

Influence of feed restriction on the wool proteome: A combined iTRAQ and fiber structural study

Seasonal weight loss is the main limitation to animal production worldwide, significantly affecting the productivity of milk, meat and wool farms, particularly in drought-prone areas of the world where most of the large-scale wool production farms are located. Although the effect of nutritional status on wool quality parameters has been extensively studied, little is known on how it affects wool protein composition. Here, a proteomic approach has been applied to study changes in fiber structure and protein composition in wool from merino sheep subjected to experimentally induced weight loss. Results indicate that there is a significant reduction in the fiber diameter of wool from the animals on a restricted diet over a 42-day period. At the same time, significant increases in the expression of the high sulfur protein KAP13.1 and proteins from the high glycine-tyrosine protein KAP6 family in the wools from the animals on the restricted diet were also detected. Such findings have strong implications for the wool industry that favors finer wool. Seasonal weight loss caused by poor pasture availability has strong effects on wool productivity parameters and quality traits. In this work we determine that experimentally induced weight loss causes a decrease in fiber diameter associated with an increase in the level of high sulfur protein KAP13.1 and proteins from the high glycine-tyrosine protein KAP6 family. The implication of this is that decreasing the fiber diameter of the wool by this process could result in a fiber reduced prickle but with reduced wearability and appearance retention.

Modeling Deamidation in Sheep α-Keratin Peptides and Application to Archeological Wool Textiles

Deamidation of glutamine (Q) and asparagine (N) has been recognized as a marker of degradation and aging in ancient proteins. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to study deamidation in wool textiles, we identified eight peptides from α-keratin proteins in sheep wool that could potentially be used to assess the level of degradation. For each chosen peptide, the extent of deamidation was determined by comparing the calculated theoretical distribution with the measured distribution using a genetic algorithm that gives the best fit to the measured distribution. Variations in the levels of deamidation were observed between peptides and in modern wool samples buried for up to 8 years in which deamidation levels were relatively low under short-term burial. In contrast, deamidation was higher in archeological textile fragments from medieval sites ranging from the 9th to 13th century in York (United Kingdom) and Newcastle (United Kingdom) and from the 13th to 16th century in Reykholt (Iceland). Major differences were observed between the British and the Icelandic samples, showing a negative correlation between age of samples and levels of deamidation, but highlighting the effect of local environment. In addition, nanoscale liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) data indicated that deamidation in wool's α-keratin was influenced by primary and higher-order structures. Predominance of deamidation on glutamine rather than asparagine in the archeological samples was attributed to a higher abundance of Q in the α-helical core domain of keratins, neighboring residues and steric hindrance preventing deamidation of N.

PH-Responsive nano-fiber membrane based on PLLA and nano wool protein for controlled release of anti-tumor drugs

pH-Responsive nano-fiber membrane based on PLLA and nano wool protein for controlled release of anti-tumor drugs

The human squamous epithelial cell envelope: the structural model by Peter M Steinert

The human squamous epithelial cell envelope: the structural model by Peter M Steinert

Wool-waste valorization: production of protein hydrolysate with high antioxidative potential by fermentation with a new keratinolytic bacterium,Bacillus pumilusA1

Wool, a recalcitrant waste mainly composed of keratin, constituted a serious problem for the environment and was not effectively valorized. This study reported the optimization of wool-waste biodegradation by a new keratinolytic bacterium Bacillus pumilus A1. The in vitro digestibility and the antioxidant potential of wool protein hydrolysate (WPH) were also investigated. The antioxidant potential of WPH was evaluated using in vitro antioxidant assays, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, reducing power and metal (Fe(2+)) chelating activity. Cultivation on 50 g l(-1) of wool for 2 days, at 45°C and at initial pH of 10, resulted in maximum production of amino acids and peptides (39.7 g l(-1)). WPH presented a very high in vitro digestibility (97%) as compared with that of the untreated wool (3%). The keratin present into the wool-waste was completely solubilized. Interestingly, WPH presented an important DPPH radical-scavenging activity with an IC50 value of 0.14 ± 0.01 mg ml(-1). WPH would be a very useful source of protein and antioxidants in animals' diets.

Preparation and Characterization of Regenerated Wool Protein PAN Blended Fibers

Wasted wool fibers were dissolved by reductive method. The dissolved wool protein was extracted by the membrane technology and blended with the Polyacrylonitrile (PAN) polymer to prepare regenerated fiber. Effects of pH value and dissolution assisting agent type on dissolution percentage of the wasted wool fibers in sodium pyrosulfite reductive system were investigated. Effects of the drawing times on properties of the fibers were also studied. Results indicated that the dissolution percentage rose with the increase of pH value of the reductive solution. Metallic salts have better dissolution assisting effect than urea. Breakage percentage of the as-spun fiber and tenacity of the regenerated protein fiber rose with the increase of the drawing times.

Unravelling the proteome of wool: Towards markers of wool quality traits

With ongoing efforts to make wool more competitive alongside other fibres, notably synthetics, there is a need to obtain a better understanding of the relationship between protein composition and characteristic wool properties to assist sheep breeding programmes. Before this can be achieved, the wool proteome needs to be mapped, by gel and non-gel techniques, and methods developed to reliably quantitate protein expression. Nevertheless, in setting out to achieve this, there are numerous challenges to be faced in the application of proteomics to wool, including the relative lack of wool protein sequence information in the publically accessible databases, the wide variety of proteins in the wool fibre, the high homology within the Type I and Type II keratins, the high degree of homology and polymorphism within individual keratin associated protein families, the dominance of the keratin proteins over others in wool and the peculiar chemistries found in keratins and their associated proteins. This review will discuss the various strategies that have been developed to both identify these proteins in the wool protein map and quantify them with the view to their application to the identification of markers for wool quality traits.

Determination and Validation of Markers for Heat-Induced Damage in Wool Proteins

Protein-based animal fibres of commercial importance are frequently exposed to elevated temperatures during processing treatments. Hydrothermal processes cause protein deterioration, impacting negatively on the value or condition of these materials. This study was designed to investigate hydrothermal damage in wool proteins at the molecular level. The effect of hydrothermal damage on Type I and II intermediate filament proteins (keratins) extracted from wool was characterised using advanced quantitative techniques based on isobaric iTRAQ labelling and mass spectrometry. Many native peptides were observed to be degraded and modified. Amongst these, twenty keratin peptides were observed to consistently degrade during hydrothermal exposure. These peptides acted as molecular markers of damage – specific indicators of the extent of heat-induced protein damage. This technology will be of value in assessing the severity of damage imparted after high temperature exposure of protein-based animal fibres such as wool and cashmere during processes such as dyeing and carbonising, or even after high temperature human hair treatments. The identification of molecular damage markers identified within wool and other materials provides a new route to sensitive and specific evaluation of the effects of protein deterioration. It is anticipated that the utilisation of such markers will facilitate the development of targeted approaches to minimising processing damage to high-value fibres and protein-based biomaterials.

Influence of different pretreatments on the accessibility of transglutaminase and tyrosinase to wool fibre proteins

Wool fibres are subject to a variety of industrial chemical treatments prior to finishing processes. The influence of such treatments on subsequent enzymatic treatments is still poorly characterised, but its importance is paramount to achieve maximal efficiency of enzymatic action on the fibres. The effect of such treatments on wool fibre structure and their significance on the accessibility of the enzymes transglutaminase and tyrosinase to their target amino acids within wool proteins was investigated. These enzymes are of great interest due to their potential as protein cross‐linkers and their capability to covalently graft beneficial compounds onto proteins. The accessibility of target amino acids was assessed using specific peptides tagged with fluorescent probes, followed by epifluorescence or confocal microscopy. Differences in the action of the different chemical pretreatments were found, with a treatment with permonosulphuric acid resulting in the greatest level accessibility to both enzymes, followed by chlorination and plasma treatments. This was confirmed by the increase in tensile strength of treated wool observed in the enzyme‐treated yarns. This work illustrates that the accessibility of these enzymes to their target aminoacids in solid substrates such as wool is key for their action and to achieve the desired property modifications. The techniques used have the advantage of permitting the visual monitoring of the diffusion of the active enzymes through the wool fibre structure and to relate this to the extent of enzymatic reaction. Crucially, this will allow an informed choice of the best wool processing stage at which to introduce such enzyme treatments.

Property and Application of Rabbit Protein Powder

Rabbit hair is a kind of animal hair, containing a large amount of keratin which can be extracted. According to the preparation method of wool protein powder, rabbit hair powder was obtained, and the morphology of the protein powder was observed by microscopy. By reacting with the alkali solution, two kinds of protein powders which were prepared from rabbit hair and wool, were used in modification of polyester fabric. Experimental results showed that samples with rabbit hair protein powder has evident improvement in moisture absorption of polyester fabric compared with untreated and wool powder treated samples, while UV protective properties of sample treated by rabbit hair powder was unexpected decreased, a further investigation will carry out to explain it.

Surface modification of wool with protease extracted polypeptides

Polypeptides were extracted from wool protein fibres using the serine type protease Esperase 8.0L (EC 3.4.21.62), a subtilisin from Bacillus sp., in a reducing solution. The extracted polypeptides, in aqueous liquor, were then applied to modify the fibre surface of wool fabric with or without additional protease. The treated wool fabric was subsequently treated with the cross-linking agent, glycerol diglycidyl ether, and then underwent a curing process to affix the polypeptide to the fibre. The resulting knitted fabric showed a very high level of shrink-resistance to machine washing, without excessive fibre damage. Shrinkage of 1-2% could be achieved after 5 times 5A washes with minimal (<1%) weight loss due to washing and a burst strength of 317 kPa.

The Physical Properties of Ultra-Short Leather Fiber Extracted from Chromed Leather Waste and its Use in Textile Industry

In order to probe the possibility of utilizing chromed leather wastes from the view of textile, firstly ultra-short leather fiber of 0.01～0.5cm was extracted by special equipment. Then the fiber structure was characterized; the moisture absorbency was tested and compared with wool and silk protein fibers. On the above basis, ultra-short collagen fiber suede fabric was developed successfully. The results indicate that ultra-short collagen fibers exist in bundles, adhesion forces between fibers are larger those of single (bundle) fiber strength. Their moisture absorbency is similar to that of wool. The micro-suede fabric made is suitable for decorating materials.

Structural analysis of alpha-helical proteins from wool using cysteine labelling and mass spectrometry

A simple reduction/labelling/extraction protocol has been developed to fractionate cortical matrix proteins from filament proteins in wool. Through differential labelling of cysteine residues their relative accessibility in the wool fibre has been investigated. This has allowed the preliminary development of a map of the chemical functionality that is accessible within wool fibres under native conditions. Protein analyses of wool subjected to mechanical action, wet chemical permonosulphate/sulphite treatment and dry argon plasma treatment revealed that none of these detectably improved the accessibility of functional groups at the wool cortex. It is anticipated that this analytical method can be extended to improve the sensitivity and scope with which chemical functionality within native fibres can be mapped and lead to a better understanding of the potential limits/opportunities for fibre modification.

Water sorption of nails treated with wool keratin proteins and peptides

Water has a considerable effect on human keratin fibres, such as nails, and is therefore crucial for their cosmetic performance. Wool proteins are mild, natural, biodegradable and are sustainably produced with multiple functionalities. They have a potential for use in the cosmetic and detergent markets. The effectiveness of two wool keratin ingredients in restoring the water sorption characteristics of nails was determined. Acetone treatment modified nail water sorption, resulting in an increase in water sorption capacity and in nail permeability. The application of keratin peptides and proteins to healthy and damaged nails improved water sorption properties, reducing permeability, especially in the case of wool keratin protein treatment.

Studies on the remedial effect of transglutaminase on protease anti‐felting treated wool

Oxidative protease treatment can improve anti‐felting of wool and also result in its loss of strength. In this study, one kind of microbial transglutaminase (MTG) was applied in the wet process of wool fabrics. The results indicated that transglutaminase could remediate wool damage following hydrogen peroxide and protease anti‐felting finishing, resulting in an increase in wool fabric strength and a decrease in alkali solubility. In addition, the evidence was provided that some cross‐linking through transglutaminase had occurred in the wool protein. The SEM showed that the scale structure became smoother after MTG treating. Analyzing of the thermal behavior revealed that the thermal stability of the MTG‐treated wool was enhanced. UV‐spectrum analysis suggested a decrease in protein extracted from the intercellular cements of the treated wool with MTG.

Photochemical crosslinking of soluble wool keratins produces a mechanically stable biomaterial that supports cell adhesion and proliferation

Keratins extracted from various "hard tissues" such as wool, hair, and nails are increasingly being investigated as a source of abundant, biocompatible materials. In this study we explored a recent photochemical method to crosslink solubilized wool keratoses, with the aim of producing a mechanically favorable biomaterial. Wool proteins were isolated by oxidizing the disulfides and extracting the resulting soluble keratoses. The α- and γ-keratose fractions were analyzed by liquid chromatography-mass spectrometry to identify their constituent proteins. Hydrogels were produced by covalent crosslinking of the α-keratoses via a photo-oxidative process catalyzed by blue light, a ruthenium complex, and persulfate. The presence of dityrosine crosslinks was demonstrated by high performance liquid chromatography and mass spectrometry analyses. The crosslinked α-keratose material had moderate tensile strength and elasticity, and high adhesive strength. The material displayed modest shrinking after crosslinking, however the shrinking could be prevented by crosslinking in the presence of 2.5% glycerol, resulting in gels that did not shrink or swell. Small solutes such as Tris and glycerol influenced the crosslink density and elastic modulus of the crosslinked material. The α-keratose was able to support adhesion and growth of NIH/3T3 fibroblasts in vitro. The fabrication of mechanically stable keratin biomaterials by this facile photo-crosslinking method may be useful for various tissue engineering applications.

Electrophoretic mapping of highly homologous keratins: A novel marker peptide approach

Identification of the intermediate filament proteins (IFPs) in the wool proteome has formerly been hampered by limited sequence information, the high degree of IFP homology and their close proximity on 2-DE maps. This has been partially rectified by the recent acquisition of four new Type I and two Type II wool IFP sequences. Among closely migrating proteins, such as IFP clusters in a 2-DE map, proteins with higher sequence coverage will be assigned higher scores, but the identification of unique peptides in such tight clusters may distinguish these closely migrating proteins. Two approaches were adopted for the study of wool IFPs. In the first, searches were conducted for peptides known to be unique to each member of the family in each spot. In the second, MALDI imaging was employed to examine peptides bound to a PVDF membrane from a poorly resolved part of the Type I IFP region of the 2-DE map. As a result, a distinct picture has emerged of the distribution of the six Type I and four Type II IFPs across the 2-DE wool protein map.

Characterisation of low abundance wool proteins through novel differential extraction techniques

Fibres from human hair and wool are characterised by two main types of proteins: intermediate filament proteins (IFPs) and keratin associated proteins (KAPs). The IFPs, comprising over 50% of the fibre, tend to dominate 2-D electrophoretic maps, hindering identification of the less-abundant KAPs. This has been compounded in wool fibres by the relatively limited amount of sequence information available, with approximately 35 distinct protein sequences from ten KAP families being available, in contrast to human hair, where the sequences from well over 80 proteins from 26 KAP families are known. Additional complications include the high degree of homology within these families, ranging from 70 to 95%, and the dominance of cysteine residues in a number of KAP families with their high propensity to form cross-links. The lack of sequence information for wool KAPs has been partly overcome through the recent acquisition of new sequences. Fractionation of the proteins on the basis of their solubility with pH, urea and DTT concentration has resulted in protein extracts in which the IFP concentration has been considerably reduced. These improvements have enabled the identification of low-abundance proteins in 2-D electrophoretic maps and represent a significant advance in our knowledge of the wool proteome.

Developing the wool proteome

The wool proteome has been largely uncharted due to a lack of database coverage, poor protein extractability and dynamic range issues. Yet, investigating correlations between wool physical properties and protein content, or characterising UV-, heat- or processing-induced protein damage requires the availability of an identifiable and identified proteome. In this study we have achieved unprecedented wool proteome identification through a strategy of comprehensive data acquisition, iterative protein identification/validation and concurrent augmentation of the sequence database. Data acquisition comprised a range of different hyphenated MS techniques including LC-MS/MS, LC-MALDI, 2D-LC-MS/MS and SDS-PAGE LC-MS. Using iterative searching of databases and search result combination using ProteinScape, a systematic expansion of identifiable proteins in the sequence database was achieved. This was followed by extensive validation and rationalisation of the protein identifications. In total, 72 complete and 30 partial ovine-specific protein sequences were added to the database, and 113 wool proteins were identified. Enhanced access to ovine-specific protein identification and characterisation will facilitate all wool fibre protein chemistry and proteomics research.