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DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to [email protected]
Project title
Isolation, and Expression in Plants, of Novel Spider Silk Genes
DEFRA project code
NF0511
Contractor organisation
and location
Department of Biology, University of York
PO Box 373
York, YO10 5YW
Total DEFRA project costs
£
Project end date
Project start date
31/12/03
Executive summary (maximum 2 sides A4)
Spider silks exhibit remarkable physical characteristics and there is extensive industrial interest in using these fibres. For
example, spider dragline silk is several times tougher than Kevlar, the toughest man-made fibre. Because of their
impressive qualities, there has been a high level of interest in developing means to exploit these materials for use in high
performance materials. Toward such ends there has been considerable investment, particularly in the USA, from
companies such as Dupont and from the Defence Department. Whilst the material properties of spider silks and some
aspects of the molecular basis of these properties are now better understood, the exploitation of these materials remains
unrealised due the inability to produce them in industrial quantities. There have been attempts to farm spiders for their
silks (rather as silk moth larvae are farmed), but their lifestyle makes this highly impractical. The clear alternative to
farming spiders is to produce the silks synthetically. One approach to this is to produce the proteins from which the silks
are made by peptide synthesis and then spin these proteins to form fibres. Unfortunately, the molecular structure of the
silk proteins is too complex to allow cost effective in-vitro synthesis. The alternative approach is to transfer the genes
that encode the silk proteins into another, more amenable organism, in order to produce large quantities of protein that
can then be spun into fibres. This approach has seen a lot of research in the last two decades.
Early attempts were made to produce spider silk protein analogues in transgenic bacteria expressing either fragments of
spider silk genes or expressing synthetic genes designed to produce spider silk like proteins. A key feature of silk
proteins in general is that they are composed of repeating blocks of peptide motifs. In spider silks the repeat motifs are
long and complex essentially involving alternating blocks of stiff and elastic stretches of proteins and these give rise to
the characteristic strength and elasticity of the final fibres. Such long and repetitive proteins are extremely difficult to
produce in microbes and after many years of efforts attempts to produce these proteins in bacteria and yeast were
abandoned. The alternative approach is to produce recombinant spider silk proteins in higher eukaryotic organisms,
which generally possess the capability to make long repetitive proteins. A dramatic development in the last few years has
been the production of recombinant spider silk proteins in transgenic animals. This approach has been successfully
developed by Nexia Biotechnologies, a Canadian company http://nexiabiotech.com). Nexia produces recombinant silk
proteins in the milk of transgenic goats and then isolate these proteins to spin into fibres. Although silks have been
CSG 15 (Rev. 6/02)
1
Project
title
Isolation, and Expression in Plants, of Novel Spider Silk
Genes
DEFRA
project code
NF0511
produced successfully in this way, the production costs are extremely prohibitive and this is unlikely to serve as the
foundation for mass production.
The use of transgenic plants for the bulk production of industrial proteins is highly attractive. Plants have the obvious
advantage of production costs as compared to transgenic animals and even bacterial production because there are
photosynthetic organisms that can be grown very cheaply on a large scale and already serve as sources of industrial
proteins. We therefore proposed to use transgenic tobacco plants for the production of recombinant silk proteins.
Tobacco was our plant of choice because it is a non-food crop and has no wild relatives in the UK making the spread of
transgenes extremely unlikely should it be approved of as a crop.
Because there has been so much work in the area of spider silks, all the known silk proteins are already largely protected
by patents and we therefore decided it was important for us to establish our own IP position in the area if the project were
to lead to the production of a valuable crop for UK agriculture and industry. To this end we proposed to clone a novel
spider silk gene free from existing IP protection. Our choice of spider from which to clone the silk gene was carefully
considered. Our colleague, Professor Fritz Vollrath at Oxford, had carried out a survey of the mechanical properties of
silks from a range of different spiders and identified that the African Nursery Web Spider (Euprosthenops Sp.) produced a
silk that was stronger, stiffer and less sensitive to water and other solvents than those of other spiders. Because of the
superior quality of its dragline silk as well as the fact that, at the start of the project, no genes had ever been cloned from
Euprosthenops, we decided to focus on this species. The complication to using this species was that we would need to
obtain wild specimens, which had to be gathered in Kenya and shipped to the UK.
The project began in October 2000, but unfortunately, due to an unforeseen scarcity in the field, our sub-contractor was
unable to deliver the spiders to us until May the following year. Although this delay immediately put the programme
several months behind schedule, we made good use of the time available to gather some important molecular data related
to the process by which the silk proteins are spun into high-performance fibres in spiders. This work was carried out
using Nephila senegalensis, a large tropical species much worked on with regard to silk properties. We made several
cDNA libraries using RNA specific to different regions along the silk gland and spinneret and sequencing several
hundred genes from these libraries allowed us to identify a number of important proteins involved in silk processing.
This work led to the filing of a potentially valuable patent in the area of silk fibre cross-linking as well as a published
paper in an international journal.
Once we obtained the spiders in May, we managed to partially clone the dragline silk protein-encoding gene that was the
target of our work within the year. This was an exciting time for our research because we rapidly discovered that the
sequence of the protein encoded by this silk gene indicated the molecular basis of the superior qualities of Euprosthenops
silk. We have filed a patent protecting the novel sequence of this gene and its expression in transgenic plants and have
published a paper regarding the sequence of this silk protein with relation to the mechanical properties of the silk.
The cloning of the Euprosthenops silk gene paved the way for its eventual expression in transgenic plants. However, at
this point an unexpected event led us to change the direction of the project. In June 2001 a German group published a
paper detailing the expression of a previously characterised silk protein gene in tobacco and potato plants. This group
used methods to produce the recombinant proteins in plants that were essentially identical to our proposed approach.
That is to say that the spider silk protein gene was expressed under the control of a constitutive promoter through
transformation of the tobacco nuclear genome. Whilst this publication clearly invalidated the novelty of our approach and
thus severely impaired our potential IP position and thus value of our work, it also revealed to us the shortcomings of this
approach. Whilst these researchers showed that the expression strategy certainly led to the production of recombinant silk
proteins, the levels produce were not of sufficient magnitude to suggest that it would be cost effective. At around the
same time some groundbreaking work was published that showed that extremely high levels of recombinant protein could
be produced in transgenic tobacco where the chloroplast genome (rather than the nuclear genome as is usually used) was
the site of transformation and gene expression. Each cell in a plant leaf contains about 100 chloroplasts and each
chloroplast contains many copies of its genome, whilst in contrast each cell typically contains two copies only of the
nuclear genome. After discussing this with DEFRA officials it was agreed that a chloroplast expression strategy with our
silk gene held more potential rather than following the original plan, which had just been scooped.
Chloroplast transformation is a new scientific area and one we had no experience with. The single publication showing
extremely high expression levels cam from a group working in The University of Florida and I immediately contacted the
head of this group to request their expression vector. On the telephone, this person was extremely friendly and helpful
and agreed to help us by providing both the vector and with technical assistance by phone and e-mail. Unfortunately,
delivery of the vector was another matter and was subject to continual complications and delay caused apparently by
CSG 15 (Rev. 6/02)
2
Project
title
Isolation, and Expression in Plants, of Novel Spider Silk
Genes
DEFRA
project code
NF0511
technical problems and then over protracted wrangling over intellectual property rights. We eventually received the
expression vector in April of the final year. In the intervening time, the RA used DNA cloning to engineer a construct of
our silk gene suitable for expression from this vector. After discussion with a DEFRA official, we were given an
unfunded extension of the project until December 2003 to allow us to produce transgenic plants.
Cloning our expression construct into the expression vector was challenging on two counts. Firstly, like all silk genes
that from Euprosthenops has a highly repetitive sequence and this is readily recombined during cloning procedures in
bacteria resulting in aberrant constructs. Secondly, the suppliers of the vector refused to supply us with even a
rudimentary map (apparently because of IP restrictions) making it very difficult for us to refine our cloning strategy.
Eventually a suitable expression construct was produced and verified by DNA sequencing in October 2003. The
construct was introduced to tobacco leaves by bombardment with DNA-coated gold particles. The regeneration of plants
from this transformation procedure requires several months of tissue culture. The first generation of plants produced are
very often not homoplastomic (they possess a mix of transformed and wild type chloroplasts) and it is necessary to pass
material from these plants through a second round of tissue culture in selective media to produce high level expressers.
After discussion with DEFRA a final unfunded extension of the project was granted to allow us to produce transgenic
plants for analysis. We have now verified the presence of the silk gene in regenerated first generation plants by PCR
methods and are in the process of producing the second generation, which are predicted to have higher expression levels.
CSG 15 (Rev. 6/02)
3
Project
title
Isolation, and Expression in Plants, of Novel Spider Silk
Genes
DEFRA
project code
Scientific report (maximum 20 sides A4)
CSG 15 (Rev. 6/02)
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NF0511