BIOMIMETIC MANUFACTURING OF FIBERS Michael Ellison (leader), Albert Abbott (co-pi), Florence Tuelé

Transcription

1 M98C-05-1 BIOMIMETIC MANUFACTURING OF FIBERS Michael Ellison (leader), Albert Abbott (co-pi), Florence Tuelé Goal Statement Our goals for this seed project were to identify the fundamental molecular biology required for the clonal production of fiber forming protein polymer through genetic expression in plants study arachnid biology for silk production and lay the foundation for the application of that biology to protein fiber wet spinning. Abstract Our research program is de facto divided into two thrusts. In the molecular biology work, we have utilized published sequences of spidroin 1 and 2 genes to construct oligonucleotides which have been linked together to build repetitive gene units for protein expression studies. The structure of the protein we are using as a model is, of course, that of spider silk, in particular, the silk used for dragline. The other major effort is the design of a spinning system that is modeled after the clearly successful activities of spiders. In order better to understand the design criteria for a protein spinning system, a study of spider biology was undertaken, guided by the vision of a system constructed of flexible, intelligent materials. Construction of Synthetic Spider Silk Genes Production of β sheet proteins We have utilized published sequences of spidroin 1 and 2 genes to construct oligonucleotides which have been linked together to build repetitive gene units for protein expression studies (Figure 1). We are currently linking these initial repetitive units to build genes encoding longer homopolymer and heteropolymer repeats.

2 M98C-05-2

3 M98C-05-3 We are in the process of moving the initial repetitive sequence units into expression systems. We are concentrating on yeast and higher plants. In the case of yeast, we will use the Pichia pastoris system (Invitrogen) as this has been shown by others to give substantial yields of recombinant protein and can be used to quickly verify that our constructs are making the appropriate proteins (Fahnestock and Bedzyk, 1997). For plant expression studies, we have begun the process of making the transgene constructs for introducing the polymer genes into appropriate plant hosts. In cooperation with Agra Tech Seeds Inc., we are investigating the use of several seed specific promoters under characterization in the Abbott laboratory to test the feasibility of expressing these synthetic genes in the seeds of legumes, particularly the peanut. We also plan to investigate the use of tobacco, as this is a model for plant transgenic production, and rice. Production of helical proteins We are investigating the large-scale production of helical protein polymers by examining the feasibility of expressing collagen genes in plants. We have available, through our collaborators in related projects, cloned collagen genes that we propose to express in yeast and in plants to obtain sufficient protein for fiber spinning studies. This work is in the planning stages. Biology of the Spider Spinning System In order better to understand the design criteria for a protein spinning system, a study of spider biology was undertaken, guided by the vision of a system constructed of flexible, intelligent materials. To this end, we enlisted the support of an expert in the field of spider spinneret biology, Dr. Jacqueline Palmer, with whom we have entered into a consulting agreement. The following description of spider anatomy and function has been adapted from one of her reports to the research team at Clemson. The material is partially taken from two of her publications, from which the figures are taken (J. M. Palmer, 1982; J. M. Palmer, 1985). We have also included additional references. For an extensive review of the spider silk literature per se, see the report by Steve Warner, et al., in the 1996 NTC Annual Report (G95-08). External Spiders have 2 body parts (abdomen and cephalothorax), 8 legs, 2 pedipalpi, 2 chelicerae (pedipalpi with chelicerae are feeding appendages) all located on the cephalothorax (Fig. 2, from R. A. Foelix, 1996). Most spiders have 3 pairs of spinnerets located on the ventral (belly)

4 M98C-05-4 Figure 3 spinneret and the shaft of the spigot, while other spigots appear as one continuous piece. Some spigots appear to have articulations external to the spinneret. (Figure 4). The ratio of the length to the diameter of the duct within the part of their abdomen. Primitive spiders had 4 pairs of spinnerets but in most evolved spiders, the anterior median pair is lost or greatly modified. Spinnerets have tiny hair-like spigots on them through which the silk fibers exit. In Tarantulas and Trapdoor spiders, the spigots, which can be very long, are found all along the ventral surface of the spinnerets. In other, more highly evolved spiders the spigots seem to be concentrated on the tips of the spinnerets and are usually short and stubby (Fig. 3). Spigots come in a wide variety of forms; some are long, some short. Some spigots are articulated with a distinct base at the junction of the Figure 4a spigot used for dragline silk production is in the approximate range of 8 to 10. Overall, spigot L/D ratios can range from 2 to 100. (H. Peters, 1955; J. Kovoor, 1986) Figure 4b. Scale bar is 10 microns Internal Each spigot is served by an individual silk gland. Some primitive spiders have only one type of spigot and only one type of general-purpose silk, produced by a single type of gland. Silk glands are connected to their spigots by way of very fine ducts, some with valve mechanisms and some without (Fig. 6). Normally, several ducts will be grouped together presumably for mechanical integrity. Silk glands are usually composed of several secretory regions producing a silk with a bicomponent sheath/core

5 M98C-05-5 structure (Fig. 7). Sometimes there are more than two secretory regions in a gland, resulting in the production of a concentric laminar filament. Sometimes these materials are proteinaceous, while in Figure 6 (from Foelix, 1996) other instances they contain a significant carbohydrate component. Different types of silk glands are used to make different types of silk that have distinct properties and are used for specific purposes. Some examples are: Egg Cases - Aciniform Glands (PMS) Cocoons - Tubuliform Gland (PMS) Swathing Silk - Aciniform Gland (PMS) Sperm Web - Aciniform Gland (PMS) Attachment Disc. - Piriform Gland (ALS) *Drag Lines - Ampullate Gland (ALS) Sticky Spiral of Web- Aggregate Gland (PLS) Sticky Spiral Support- Flagilliform Gland (PLS) *Web Frame Line - Ampullate Gland (ALS) (A= Anterior; L=Lateral; M=Median; P=Posterior; S=Spinneret) In addition to the varied kinds of glands and silks, spiders sometimes combine silks to produce threads with composite properties. Almost all spider silk research fueled by a textile Figure 7 fiber interest has been focused on ampullate gland silk (*), the material used for draglines. This silk has the most remarkable mechanical properties of all the silks, and is easiest to obtain for study. The silk material is stored in the gland prior to spinning. In some instances it is stored intracellularly while in others the material is stored in the gland lumen. There is some evidence of sulfhydryl and disulfide bonding intracellularly that has disappeared upon secretion of the silk. The relevance of this observation may lie in answering the important question of stability of the stored silk material to solidification in vivo. Furthermore, levels of structural orientation such as peptide bond formation between separate proteins and subsequent organization, even polymerization, into fibers, must be controlled prior to spinning. The silk in the gland does not display any birefringence, but birefringence develops as the silk passes down the duct, the structure apparently being a consequence of mechanical forces on the silk (M. Hinman, 1992). The structural feature that offers the most promise for exploitation

6 M98C-05-6 is the liquid crystalline (LC) nature of spider silk under appropriate conditions (C. Viney, 1992). In that same paper, Viney reported the results of a study of the transition in spider silk from isotropic to nematic LC optical structure. One result is that the nematic structure is concentration dependent. For the continuing work on the spinning side of the project, we are going to determine the best way to exploit the LC phase transition. Our earlier design concept, a labile tag on one end of the protein chain to impart a flow-induced orientation, will be supplemented with a concentration gradient in the duct (capillary) to induce the LC transition. In this way, we expect to facilitate the filament formation. In addition, we plan to design biology experiments on the solidification of the silk in vivo. References Fahnestock, S.R. and L.A. Bedzyk Production of synthetic spider dragline silk protein in Pichia pastoris. Appl. Microbiol. Biotechnol. 47: Foelix, R. A "Biology of Spiders, 2nd ed." Oxford University Press. Hinman, M., Z. Dong, M. Xu, R. V. Lewis, Spider Silk: A Mystery Starting to Unravel, in "Results and Problems in Cell Differentiation 19: Biopolymers," S. T. Case, ed., Chapter 8. Kovoor, J <title>, Rev. Arachnologie, 7, 15. Peters, H., <title>, Zoo. Naturforsch., 10B, 395. Palmer, J. M., F. A. Coyle and F. W. Harrison, Structure and Cytochemistry of the Silk Glands of the Mygalomorph Spider Antrodiaetus unicolor (Araneae, Antrodiaetidae), J. Morph 174, 269. Palmer, J. M The Silk and Silk Production System of the Funnel-Web Mygalomorph Spider Euagrus (Araneae, Dipluridae), J. Morph, 186, 195. Viney, C., The Nature and Role of Liquid Crystalline Order in Silk Secretions, in "Results and Problems in Cell Differentiation 19: Biopolymers," S. T. Case, ed., Chapter 9.