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1 Framingham State University Digital Commons at Framingham State University Biology Department Faculty Publications Biology Department 1994 Sequence Conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae) Richard D. Beckwitt Framingham State University, Steven Arcidiacono Follow this and additional works at: Part of the Biology Commons Citation Beckwitt, Richard D. and Arcidiacono, Steven. "Sequence Conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae)." Journal of Biological Chemistry 269 (1994): Accessed at This Article is brought to you for free and open access by the Biology Department at Digital Commons at Framingham State University. It has been accepted for inclusion in Biology Department Faculty Publications by an authorized administrator of Digital Commons at Framingham State University. For more information, please contact

2 Journal of Biological Chemistry (1994) 269 xxx- xxx Accepted November 15, 1993 SEQUENCE CONSERVATION IN THE C- TERMINAL REGION OF SPIDER SILK PROTEINS (SPIDROIN) FROM Nephila clavipes (TETRAGNATHIDAE) AND Araneus bicentenarius (ARANEIDAE) Running title: Sequence conservation in spider silk proteins Richard Beckwitt Department of Biology Framingham State College Framingham, MA (508) and Steven Arcidiacono 1 Biotechnology Division U.S. Army, Natick Research, Development and Engineering Center Natick, MA (508) FAX: (508) To whom correspondence should be addressed.

3 SUMMARY T h e P o l y m e r a s e C h a i n R e a c t i o n ( P C R ) h a s b e e n u s e d t o a m p l i f y t h e portion of the Spidroin 1 gene that codes for the C- terminal part of the silk protein of the spider Nephila clavipes. Along with some substitution mutations of minor consequence, the PCR- derived sequence reveals an additional base missing from the previously published Nephila Spidroin 1 sequence. Comparison of the PCR- derived sequence with the equivalent region of Spidroin 2 indicates that the insertion of this single base results in greatly increased similarity in the resulting amino acid sequences of Spidroin 1 and Spidroin 2 (75% over 97 amino acids). The same PCR primers also amplified a fragment of the same length from Araneus bicentenarius. This sequence is also very similar to Spidroin 1 of Nephila (71% over 238 bases excluding the PCR primers, which translates into 76% over 79 amino acids). 1

4 Spiders have the ability to synthesize several different types o f s i l k. A l t h o u g h r e l a t e d, t h e s e s i l k s h a v e b e e n s h o w n t o d i f f e r i n t h e i r a m i n o a c i d c o m p o s i t i o n a n d p h y s i c a l p r o p e r t i e s. C o n s i d e r a b l e r e c e n t i n t e r e s t h a s b e e n c o n c e n t r a t e d o n t h e d r a g l i n e s i l k o f Nephila clavipes (L.)(Kaplan et al. 1992). Xu and Lewis (1990) and Hinman a n d L e w i s ( ) p u b l i s h e d t h e p a r t i a l c D N A s e q u e n c e s o f t w o g e n e s, Spidroin 1 and Spidroin 2, both thought to be active in the major ampullate gland, and both thought to be components of dragline silk. Although the two genes appear to be distinct, they do share some similarities, both in overall organization, and in some regions of apparent sequence conservation. When translated into amino acid sequences, the bulk of each polypeptide consists of highly repeated domains. Both polypeptides have regions of 5-8 alanines within larger repeating units. In Spidroin 1 the repeats are often GAGQGGYGGLGGQ, while in Spidroin 2 the repeats are often GPGGYGPGQQ. I n a d d i t i o n, t h e C - terminal portions of the two polypeptides are very similar: Hinman and Lewis (1992) report 80% similarity over a 49- amino acid region. Mello et al. (1993) report amino acid composition and partial peptide sequencing of both the dragline fiber a n d t h e c o n t e n t s o f t h e m a j o r a m p u l l a t e g l a n d o f N e p h i l a. T h e a m i n o acid composition data and peptide sequences are the same as those predicted from the cdna sequence of Spidroin 1, b u t n o t o f S p i d r o i n 2. I t i s p o s s i b l e t h a t S p i d r o i n 2 r e p r e s e n t s t h e p r o d u c t o f a s i l k g e n e t h a t i s a c t i v e i n a d i f f e r e n t g l a n d, a n d i s n o t a c o m p o n e n t o f the dragline silk. 2

5 A s p a r t o f o n g o i n g e f f o r t s t o i n v e s t i g a t e t h e m o l e c u l a r b i o l o g y o f s p i d e r s i l k, w e h a v e m a d e u s e o f t h e p u b l i s h e d s e q u e n c e o f S p i d r o i n 1 t o d e s i g n p r i m e r s f o r P o l y m e r a s e C h a i n R e a c t i o n ( P C R ) t h a t a m p l i f y t h e 3 ' r e g i o n o f t h e g e n e. S e q u e n c i n g o f t h e P C R p r o d u c t s i n d i c a t e d t h a t t h e r e w a s a n e r r o r i n t h e p u b l i s h e d S p i d r o i n 1 s e q u e n c e, a n d t h a t a s a r e s u l t, t h e C - terminal regions of Spidroin 1 and Spidroin 2 are m u c h m o r e s i m i l a r t h a n p r e v i o u s l y t h o u g h t. I n a d d i t i o n, w e h a v e u s e d t h e s a m e p r i m e r s t o a m p l i f y t h e e q u i v a l e n t r e g i o n f r o m t h e p r e s u m e d Spidroin 1 of Araneus bicentenarius (McCook). MATERIALS AND METHODS Preparation of genomic DNA: DNA was extracted from spiders, following the method of Strauss (1987). Approximately 1 g of frozen abdominal tissue was crushed with a mortar and pestle under liquid N 2, t h e n d i g e s t e d i n 1 2 m l o f m M N a C l, 1 0 m M T r i s - H C l p H 8. 0, 2 5 m M E D T A, 0. 5 % S D S a n d 0. 1 m g / m l P r o t e i n a s e K, f o r 1 8 h o u r s a t 5 0 o C. After extraction by 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1), nucleic acids were precipitated by adding 1/2 volume of 7. 5 M a m m o n i u m a c e t a t e a n d 2 v o l u m e s o f c o l d e t h a n o l. A f t e r p e l l e t i n g t h e D N A b y c e n t r i f u g a t i o n, a n d r i n s i n g i t w i t h 7 0 % e t h a n o l, t h e D N A w a s d i s s o l v e d i n 1. 5 m l o f 1 0 m M T r i s - H C l p H 8. 0, 1 m M E D T A ( T E ). D N A f r o m t h e s e p r e p a r a t i o n s w a s o f h i g h m o l e c u l a r w e i g h t ( > 2 0 k b p ) w h e n examined by gel electrophoresis. Polymerase Chain Reaction: Primer sequences were taken from Xu a n d L e w i s ( ), a n d s p a n a r e g i o n o f b p ( e x c l u d i n g a d d e d EcoRI 3

6 restriction sites) beginning at base 1954 of the Spidroin 1 sequence. The sequences are: S1LEFT: 5'-CGCGAATTCGGTTGTCTTCTCCTCAAGCTAGTTC-3' S1RIGHT: 5'-GGCGAATTCCCTAGGGCTTGATAAACTGATTGAC-3'. P C R w a s c a r r i e d o u t f o l l o w i n g t h e m e t h o d s o u t l i n e d i n S a i k i e t a l. (1988): Templates for each reaction were diluted to appropriate concentration in 1 μl o f H 2 O. A P C R r e a c t i o n c o c k t a i l s u f f i c i e n t f o r the planned number of reactions was mixed, consisting of (per reaction): 10 μl 1 0 X P C R b u f f e r ( m M K C l, m M T r i s - H C l p H 8. 4 a t R. T., 1 5 m M M g C l 2, 1 m g / m l g e l a t i n ), 1 0 μl 1 0 X d N T P s t o c k ( 2 m M e a c h o f d A T P, d C T P, d G T P, d T T P, p H 7. 0 ), 1 0 μl o f e a c h p r i m e r ( 2. 5 μm i n 1 0 m M T r i s - H C l p H 8. 0, 0. 1 m M E D T A ), 6 0 μl H 2 O. A n a l i q u o t o f 100 μl w a s a d d e d t o e a c h P C R r e a c t i o n t u b e, b e f o r e t e m p l a t e D N A w a s added. A negative control, consisting of all reaction components e x c e p t t e m p l a t e D N A w a s i n c l u d e d i n e a c h s e t o f r e a c t i o n s. A f t e r addition of the DNA template (1 μg of genomic DNA or 10 pg of gel- purified PCR product), each reaction was overlain with 2 drops o f m i n e r a l o i l, a n d denatured at 94 o C f o r 5 m i n u t e s. Taq polymerase ( 2. 5 u n i t s, P e r k i n - Elmer/Cetus) was then added to each reaction ("hot start"), which was then subjected to 25 cycles of the following temperature regimes: (1) Genomic DNA: denaturing at 94 o C for 1 minute, annealing at 60 o C (45 o C for Araneus) for 1 minute, and elongation at 72 o C for 2 minutes. (2) Re-amplification of PCR products: denaturing at 94 o C f o r 1 m i n u t e, a n n e a l i n g a n d e l o n g a t i o n at 72 o C for 2 minutes. In each case the final extension step was 4

7 increased by 5 minutes to insure full- length double- stranded products, after which the reactions were held at 4 o C u n t i l a n a l y z e d. Temperature cycling was performed on a Thermal Cycler (Perkin- Elmer/Cetus). PCR products were also re- amplified using Digoxigenin- 11- dutp (Boehringer Mannheim), to make hybridization probes (Finckh, et al. 1991). Cloning of PCR products: PCR products were digested with EcoRI as was the vector, puc18. The linearized vector was then dephosphorylated with calf intestinal alkaline phosphatase (CIAP). The insert and vector sequences were joined using T 4 DNA ligase, and used to transform competent cells of E. coli strains DH5αF' or XL- 1 Blue (Stratagene). Recombinant clones were identified by blue/white screening, as well as colony h y b r i d i z a t i o n, u s i n g t h e d i g o x i g e n i n - labeled PCR product as a probe, and the reagents a n d p r o t o c o l s o f t h e G e n i u s N o n - radioactive Labeling and Detection System (Boehringer- Mannheim). Clones containing inserts were selected for sequencing. D N A s e q u e n c i n g: Plasmid templates were prepared for sequencing using the standard mini- prep protocol of Sambrook, et al. (1989), including a PEG precipitation. Manual sequencing was done using the protocol of the Sequenase kit (U.S. Biochemical), or the dsdna Cycle S equencing System (BRL). Additional sequencing was done using the A.L.F. automated DNA sequencer (Pharmacia). A l l c o m p u t e r a n a l y s i s o f D N A a n d a m i n o a c i d s e q u e n c e s w a s d o n e with the IBM version of the DNASTAR package of computer programs 5

8 (DNASTAR, Inc.) RESULTS AND DISCUSSION P C R a m p l i f i c a t i o n o f g e n o m i c D N A f r o m Nephila clavipes produced a s i n g l e p r o d u c t o f a b o u t b p l e n g t h, c l o s e t o t h e p r e d i c t e d l e n g t h. The sequence of the cloned PCR product is given in Figure 1, and compared with the published sequence o f t h a t p o r t i o n o f S p i d r o i n 1. A s c a n b e s e e n, t h e r e a r e s e v e r a l m i n o r d i f f e r e n c e s : 4 a r e s i n g l e b a s e s u b s t i t u t i o n s, w h i l e o n e i s a n i n s e r t i o n a t b a s e o f t h e P C R sequence, corresponding to the base following 2083 in Spidroin 1. The substitutions are probably allelic variants in the Spidroin 1 gene. Although it is possible that the substitutions are the result of errors in reading the sequences or errors in replication that occurred during PCR (Gyllenstein and Ehrlich 1988), all were present i n several clones, derived from independent PCR reactions. The consequences of these substitutions are minor: two are silent, while t h e o t h e r t w o r e s u l t i n t h e s u b s t i t u t i o n o f v e r y s i m i l a r a m i n o a c i d s (leucine for valine at amino acid 662, and threonine fo r s e r i n e a t amino acid 672). Insertion mutations are much less likely to occur during PCR than substitutions. Also, the consequences of an insertion are much more drastic for the polypeptide produced. Although Spidroin 1 and Spidroin 2 are not identi c a l i n t h e 3 ' r e g i o n o f t h e c D N A s, t h e y a r e v e r y s i m i l a r. W h e n t h e D N A s e q u e n c e s a r e a l i g n e d i n t h i s r e g i o n, i n addition to numerous small regions of 1-3 b a s e s u b s t i t u t i o n s, t h e r e 6

9 a l s o a p p e a r s t o b e a s i n g l e i n s e r t i o n / d e l e t i o n b e t w e e n t h e m, a g a i n at the base following 2083 in Spidroin 1 (Figure 2). When the polypeptides coded for by the published sequences of Spidroin 1 and Spidroin 2 are examined, they are extremely similar over a 49- amino a c i d l o n g r e g i o n t h a t e n d s a t r e s i d u e o f S p i d r o i n 1 ( F i g u r e 3A.); t h i s c o r r e s p o n d s t o b a s e s i n t h e S p i d r o i n 1 c D N A. N o t e t h a t the region of DNA sequence similarity extends for a considerable length past this point. All of this suggests that the published sequence for Spidroin 1 is in error, and that the correct sequence has an additional "A" following base The consequences of this correction are striking. When the C - terminal portions of the polypeptides are compared, the region of similarity is extended by an additional 54 amino acids (Figure 3B). I t i s n o w 7 5 % o v e r 9 7 a m i n o a c i d s. A n o t h e r e f f e c t o f t h e f r a m e - s h i f t i s t o c h a n g e t h e p o s i t i o n o f t h e s t o p - c o d o n f r o m t o , t h e polypeptide is now 30 amino acid residues longer. As a result, certain structural features of the Spidroin 1 and Spi d r o i n 2 g e n e s a l s o b e c o m e m o r e s i m i l a r : b o t h n o w u s e T A A a s a s t o p c o d o n, a n d t h e spacing between the stop and the poly-a r e c o m b i n a t i o n s i t e i s n o w 7 5 bases in Spidroin 1 and 79 bases in Spidroin 2. T h e g r e a t d e g r e e o f s e q u e n c e s i m i l a r i t y b e t w e e n t h e C - terminal portions of the Spidroin 1 and Spidroin 2 polypeptides implies, not only homology between the two genes, but also a strong degree of sequence conservation, since the remaining portions of the two genes have diverged to a much greater degree. S i n c e b o t h S p i d r o i n 1 7

10 a n d S p i d r o i n 2 s h a r e t h e s a m e c o n s e r v e d C - terminal region, one might suspect that this would be a feature shared by all spider silk genes, or perhaps even by silk genes from other arthropods. There is no significant sequence similari t y t o t h e h e a v y c h a i n f i b r o i n o f t h e w a x moth, Galleria mellonella (Zurovec et al. 1992), which is the only lepidopteran silk for which there is any published DNA sequence data f r o m t h e 3 ' e n d o f t h e g e n e. N o p u b l i s h e d s e q u e n c e d a t a e x i s t f o r other silk genes from Nephila, nor from dragline silk of other spiders. Although the S1LEFT and S1RIGHT PCR primers were unable to amplify any product from Argiope aurantia (Araneidae) when the annealing temperature was lowered as far as 37 o C, a P C R p r o d u c t o f about 300 bp was amplified from Araneus bicentenarius (Araneidae) w h e n t h e a n n e a l i n g t e m p e r a t u r e w a s 4 5 o C. This PCR product has also b e e n s e q u e n c e d, a n d a l s o s h o w s c o n s i d e r a b l e s e q u e n c e s i m i l a r i t y t o Spidroin 1 from Nephila (Figure 4). A g a i n, t h e s i m i l a r i t y i s m o r e striking when the amino acid sequences are compared, as there are several silent (third- base) substitutions (Figure 5). When all t h r e e p o l y p e p t i d e s a r e c o m p a r e d, t h e y a r e i d e n t i c a l a t 5 4 o f 7 8 a m i n o acid positions. In addition, 21 of the remaining 24 amino acid positions are the same in two of the three polypeptides, and most substitutions are between amino acids of very similar characteristics (isoleucine for valine, or serine for alanine, proline or glycine). T h u s i t a p p e a r s t h a t t h e n o n - repeating C- terminal portion of spidroin is highly conserved, not only between the silk proteins of one 8

11 species, but between species as well. We are assuming that the sequence amplified from Araneus represents Spidroin 1. We also as s u m e t h a t g e n e d u p l i c a t i o n o r s o m e similar process produced the multiple silks that are typical of orb- weaving spiders before the phylogenetic event that separated the families Tetragnathidae and Araneidae. This is not obvious from a simple comparison of sequence similarities. There is 70.6% similarity between Nephila and Araneus Spidroin 1 DNA over 238 bases, w h i c h t r a n s l a t e s i n t o % s i m i l a r i t y o v e r 7 8 a m i n o a c i d s. S p i d r o i n 1 a n d S p i d r o i n 2 o f N e p h i l a h a v e a D N A s e q u e n c e s i m i l a r i t y o f % o v e r t h e s a m e b a s e s, w h i c h t r a n s l a t e s i n t o % o v e r 7 8 a m i n o acids. Since the C- terminal region of spidroin seems to be so highly conserved, naive application of any molecular clock analysis to estimate time since divergence would be inappropriate. I t r e m a i n s t o b e s e e n w h a t t h e f u n c t i o n a l s i g n i f i c a n c e o f t h i s highly conserved C- terminal portion of the protein may be. One possibility is that this portion is necessary to maintain the soluble o r l i q u i d c r y s t a l l i n e s t a t e o f s i l k w i t h i n t h e g l a n d ( K e r k a m e t a l. 1991). An examination of the amino acid composition and predicted secondary structure of this region indicates that it is distinctly different from the bulk of the repeating region of either Spidroin 1 o r S p i d r o i n 2. S o m e o f t h e s t r u c t u r a l p r o p e r t i e s a r e s u m m a r i z e d i n T a b l e 1. P r e d i c t i o n o f s e c o n d a r y s t r u c t u r e, u s i n g e i t h e r o f t h e t w o p o p u l a r m e t h o d s ( G a r n i e r e t a l , o r C h o u a n d F a s s m a n ), is not particularly satisfactory, and there is still considerable 9

12 confusion about the secondary structure of spider silk proteins. Therefore, the secondary structure analysis should not be taken as a n y i n d i c a t i o n o f t h e t r u e s t r u c t u r e. H o w e v e r, d i f f e r e n c e s i n t h e predicted structures do reveal real differences in the type and pattern of amino acids in the different peptides. This suggests t h a t, w h a t e v e r t h e s t r u c t u r e o r f u n c t i o n o f t h e C - terminal portion o f t h e S p i d r o i n p r o t e i n s i s, i t i s s o m e t h i n g d i f f e r e n t f r o m t h e r e s t of the protein. ACKNOWLEDGMENTS T h i s w o r k w a s c o n d u c t e d w h i l e R. B e c k w i t t w a s o n sabbatical in the Biotechnology Division, Natick Research, Development and Engineering Center. The financial support of the U.S. Army is gratefully acknowledged. 10

13 REFERENCES Chou, P.Y., and Fassman, G.D. (1978) Advances in Enzymology 47, Finckh, U., Lingenfelter, P.A., and Myerson, D. (1991) Biotechniques 10, Garnier, J., Osguthorpe, D., and Robson, D. (1978) J. Mol. Biol. 120, Gyllenstien, U.B., and Erlich, H.A. (1988) Proc. Natl. Acad. Sci. USA 85, Hinman, M.B., and Lewis, R.V. (1992) J. Biol. Chem. 267, Kaplan, D., Fossey, S., Mello, C., Arcidiacono, S., Senecal, K., Muller, W., Stockwell, S., Beckwitt, R., Viney, C., and Kerkam, K. (1992) M.R.S. Bulletin 17, Kerkam, K., Viney, C., Kaplan, D., and Lombardi, S. (1991) Nature 349, Mello, C., Yeung, B., Senecal, K., Vouros, P., and Kaplan, D. (1993) American Chemical Society Symposium Series 544 (In Press) Saiki, R., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239, Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11

14 Strauss, W.M. (1987) Current protocols in molecular biology, Greene Publishing Associates and Wiley-Interscience, NY Xu, M. and Lewis, R.V. (1990) Proc. Natl. Acad. Sci. USA 87, Zurovec, M., Sehnal, F., Scheller, K., and Kumaran, A.K. (1992) Insect Biochem. Molec. Biol. 22,

15 T a b l e 1. S t r u c t u r a l p r o p e r t i e s o f t h e C - terminal region of Spidroin f r o m A r a n e u s, c o m p a r e d t o t h e r e p e a t i n g ( R ) a n d C - terminal (C) regions of Spidroin 1 and Spidroin 2 from Nephila. Hydrophobicity = Hopp/Woods index, - 100X (values in parentheses = Kyte/Doolittle i n d e x, X ) ; H e l i x, E x t e n d, T u r n, C o i l = %, b a s e d o n G a r n i e r - Robson (values in parentheses based on Chou- Fassman). Values calculated using the DNASTAR program PROTEIN. Polypeptide pi Hydro- Helix Extend Turn Coil phobicity Spidroin 1 (R) (-6) 13 (27) 2 (0) 11 (73) 74 Spidroin 2 (R) (-57) 5 (28) 50 (3) 19 (72) 26 Spidroin 1 (C) (49) 7 (19) 42 (64) 15 (31) 38 Spidroin 2 (C) (73) 9 (32) 52 (61) 9 (35) 29 Araneus (C) (48) 13 (19) 48 (62) 7 (34) 32

16 FIGURE LEGENDS F i g u r e 1. P u b l i s h e d s e q u e n c e o f N e p h i l a S p i d r o i n 1 c o m p a r e d w i t h t h e sequence obtained by PCR (Nc- Sp- 1PCR, EcoR I s i t e s o m i t t e d ). N o t e t h e gap inserted into the Spidroin 1 sequence after base The alignment was made using the DNASTAR program ALIGN, with the gap penalty set to 30. Figure 2. Published sequence of Nephila Spidroin 1 compared to Nephila Spidroin 2. The regions compared are the same as in Figure 1. Figure 3. Predicted amino acid sequence of Nephila Spidroin 1 compared to Spidroin 2. A. Published sequence. B. Corrected sequence (Nc- Sp- 1PCR), amino acids numbered to correspond to published sequence. Alignments were made using the DNASTAR program AALIGN, using the standard amino acid similarity matrix: ":" = strong similarity, "." = weak similarity. Figure 4. PCR derived sequence from Araneus (Ab- Sp- 1PCR), compared to the PCR derived sequence from Nephila (Nc- Sp- 1PCR). The PCR primer sequences have been deleted from this comparison. F i g u r e 5. C o m p a r i s o n o f p r e d i c t e d a m i n o a c i d s e q u e n c e s f r o m N e p h i l a Spidroin 1 (corrected) and Spidroin 2, and Araneus Spidroin 1. The consensus sequence includes only those amino acids shared by all three

17 polypeptides.

18 10v 20v 30v 40v 50v Nc-Sp-1PCR GGTTGTCTTCTCCTCAAGCTAGTTCAAGAGTTTCATCAGCTGTTTCCAACT GGTTGTCTTCTCCTCAAGCTAGTTCAAGA TTTCATCAGCTGTTTCCAACT Spidroin 1 GGTTGTCTTCTCCTCAAGCTAGTTCAAGACTTTCATCAGCTGTTTCCAACT 1960^ 1970^ 1980^ 1990^ 2000^ 60v 70v 80v 90v 100v Nc-Sp-1PCR TGGTTGCAAGTGGTCCTACTAATTCTGCGGCCTTGTCAAGTACAATCAGT TGGTTGCAA TGGTCCTACTAATTCTGCGGCCTTGTCAAGTACAATCAGT Spidroin 1 TGGTTGCAACTGGTCCTACTAATTCTGCGGCCTTGTCAAGTACAATCAGT 2010^ 2020^ 2030^ 2040^ 2050^ 110v 120v 130v 140v 150v Nc-Sp-1PCR AATGTGGTTTCACAAATAGGCGCCAGCAATCCTGGTCTTTCTGGATGTGA A A GTGGTTTCACAAAT GGCGCCAGCA TCCTGGTCTTTCTGGATGTGA Spidroin 1 AACGTGGTTTCACAAATTGGCGCCAGCA-TCCTGGTCTTTCTGGATGTGA 2060^ 2070^ 2080^ 2090^ 2100^ 160v 170v 180v 190v 200v Nc-Sp-1PCR TGTCCTCATTCAAGCTCTTCTCGAGGTTGTTTCTGCTCTTATCCAGATCT TGTCCTCATTCAAGCTCTTCTCGAGGTTGTTTCTGCTCTTATCCAGATCT Spidroin 1 TGTCCTCATTCAAGCTCTTCTCGAGGTTGTTTCTGCTCTTATCCAGATCT 2110^ 2120^ 2130^ 2140^ 2150^ 210v 220v 230v 240v 250v Nc-Sp-1PCR TAGGTTCTTCCAGCATCGGCCAAGTTAACTATGGTTCCGCTGGACAAGCC TAGGTTCTTCCAGCATCGGCCAAGTTAACTATGGTTCCGCTGGACAAGCC Spidroin 1 TAGGTTCTTCCAGCATCGGCCAAGTTAACTATGGTTCCGCTGGACAAGCC 2160^ 2170^ 2180^ 2190^ 2200^ 260v 270v 280v Nc-Sp-1PCR ACTCAGATCGTTGGTCAATCAGTTTATCAAGCCCTAG ACTCAGATCGTTGGTCAATCAGTTTATCAAGCCCTAG Spidroin 1 ACTCAGATCGTTGGTCAATCAGTTTATCAAGCCCTAG 2210^ 2220^ 2230^ 2240^ Similarity index = %, total bases not opposite a gap = 287 Number of gaps = 1, bases opposite gaps = 1 Length of the overlapping region = 288 NUMBER OF MATCHED BASES = 283

19 Spidroin 2 Spidroin 1 Spidroin 2 Spidroin 1 Spidroin 2 Spidroin 1 Spidroin 2 Spidroin 1 Spidroin 2 Spidroin 1 Spidroin 2 Spidroin v 1610v 1620v 1630v 1640v GTCTGGCTTCTCCAGATTCAGGCGCTAGAGTTGCATCAGCTGTTTCTAACT G TG CTTCTCC A C G C AGA TT CATCAGCTGTTTC AACT GGTTGTCTTCTCCTCAAGCTAGTTCAAGACTTTCATCAGCTGTTTCCAACT 1960^ 1970^ 1980^ 1990^ 2000^ 1650v 1660v 1670v 1680v 1690v TGGTATCCAGTGGCCCAACTAGCTCTGCTGCCTTATCAAGTGTTATCAGT TGGT C A TGG CC ACTA TCTGC GCCTT TCAAGT ATCAGT TGGTTGCAACTGGTCCTACTAATTCTGCGGCCTTGTCAAGTACAATCAGT 2010^ 2020^ 2030^ 2040^ 2050^ 1700v 1710v 1720v 1730v 1740v AACGCTGTGTCTCAAATTGGCGCAAGTAATCCTGGTCTCTCTGGTTGCGA AACG GT TC CAAATTGGCGC AG ATCCTGGTCT TCTGG TG GA AACGTGGTTTCACAAATTGGCGCCAGC-ATCCTGGTCTTTCTGGATGTGA 2060^ 2070^ 2080^ 2090^ 2100^ 1750v 1760v 1770v 1780v 1790v TGTCCTCATTCAAGCTCTCTTGGAAATCGTTTCTGCTTGTGTAACCATCC TGTCCTCATTCAAGCTCT T GA T GTTTCTGCT T T ATC TGTCCTCATTCAAGCTCTTCTCGAGGTTGTTTCTGCTCTTATCCAGATCT 2110^ 2120^ 2130^ 2140^ 2150^ 1800v 1810v 1820v 1830v 1840v TTTCTTCATCCAGCATTGGTCAAGTTAATTATGGAGCGGCTTCTCAGTTC T TTC TCCAGCAT GG CAAGTTAA TATGG C GCT CA C TAGGTTCTTCCAGCATCGGCCAAGTTAACTATGGTTCCGCTGGACAAGCC 2160^ 2170^ 2180^ 2190^ 2200^ 1850v 1860v 1870v 1880v GCCCAAGTTGTCGGCCAATCTGTTTTGAGTGCATTTT C CA T GT GG CAATC GTTT GC T ACTCAGATCGTTGGTCAATCAGTTTATCAAGCCCTAG 2210^ 2220^ 2230^ 2240^ Similarity index = %, total bases not opposite a gap = 287 Number of gaps = 1, bases opposite gaps = 1 Length of the overlapping region = 288 NUMBER OF MATCHED BASES = 203

20 A. Published sequence for Spidroin 1 660v 670v 680v 690v 700v Spidroin 1 LSSPQASSRLSSAVSNLVATGPTNSAALSSTISNVVSQIGASILVFLDVMS L:SP::::R::SAVSNLV::GPT:SAALSS.ISN.VSQIGAS : : Spidroin 2 LASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDV 540^ 550^ 560^ 570^ 580^ 710v Spidroin 1 SFKLFSRLFLLLSRSX :: : :. Spidroin 2 LIQALLEIVSACVTILSSSSIGQVNYGAASQFAQVVGQSVLSAFX 590^ 600^ 610^ 620^ B. Corrected sequence for Spidroin 1 660v 670v 680v 690v 700v Nc-Sp-1PCR LSSPQASSRVSSAVSNLVASGPTNSAALSSTISNVVSQIGASNPGLSGCDV L:SP::::RV:SAVSNLV:SGPT:SAALSS.ISN.VSQIGASNPGLSGCDV Spidroin 2 LASPDSGARVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDV 540^ 550^ 560^ 570^ 580^ 710v 720v 730v 740v Nc-Sp-1PCR LIQALLEVVSALIQILGSSSIGQVNYGSAGQATQIVGQSVYQALGX LIQALLE:VSA : IL:SSSIGQVNYG:A:Q :Q:VGQSV A: Spidroin 2 LIQALLEIVSACVTILSSSSIGQVNYGAASQFAQVVGQSVLSAFX 590^ 600^ 610^ 620^

21 10v 20v 30v 40v 50v Nc-Sp-1PCR AAGAGTTTCATCAGCTGTTTCCAACTTGGTTGCAAGTGGTCCTACTAATTC AGAGTTTCATC GCTGT TC TTGGT C AGTGG CCTACTA T C Ab-Sp-1PCR TAGAGTTTCATCGGCTGTATCATCTTTGGTATCTAGTGGACCTACTACTCC 10^ 20^ 30^ 40^ 50^ 60v 70v 80v 90v 100v Nc-Sp-1PCR TGCGGCCTTGTCAAGTACAATCAGTAATGTGGTTTCACAAATAGGCGCCA GC GC T TC A TAC ATCAGTA G GT TC CAAAT G GC A Ab-Sp-1PCR AGCCGCACTTTCTAATACTATCAGTAGCGCTGTATCGCAAATCAGTGCAA 60^ 70^ 80^ 90^ 100^ 110v 120v 130v 140v 150v Nc-Sp-1PCR GCAATCCTGGTCTTTCTGGATGTGATGTCCTCATTCAAGCTCTTCTCGAG G AATCCTGGTCTTTCTGG TG GATGT CT T CAAGCT T T GA Ab-Sp-1PCR GTAATCCTGGTCTTTCTGGTTGCGATGTACTTGTGCAAGCTTTGTTGGAA 110^ 120^ 130^ 140^ 150^ 160v 170v 180v 190v 200v Nc-Sp-1PCR GTTGTTTCTGCTCTTATCCAGATCTTAGGTTCTTCCAGCATCGGCCAAGT GTTGT TC GC CT TCCA ATC T GG TCTTC AG TCGG CAA T Ab-Sp-1PCR GTTGTATCGGCCCTCGTCCATATCCTTGGCTCTTCTAGTGTCGGGCAAAT 160^ 170^ 180^ 190^ 200^ 210v 220v 230v Nc-Sp-1PCR TAACTATGGTTCCGCTGGACAAGCCACTCAGATCGTT TAACTATGGT CC CTG CA C CA AT GT Ab-Sp-1PCR TAACTATGGTGCCTCTGCTCAGTATGCCCAAATGGTA 210^ 220^ 230^ Similarity index = %, total bases not opposite a gap = 238 Number of gaps = 0, bases opposite gaps = 0 Length of the overlapping region = 238 NUMBER OF MATCHED BASES = 168

22 Consensus RV:SAVS.LV:SGPT.:AALS..IS..VSQI:ASNPGLSGCDVL:QALLE: Ab-Sp-1PCR RVSSAVSSLVSSGPTTPAALSNTISSAVSQISASNPGLSGCDVLVQALLEV Spidroin 1 RVSSAVSNLVASGPTNSAALSSTISNVVSQIGASNPGLSGCDVLIQALLEV Spidroin 2 RVASAVSNLVSSGPTSSAALSSVISNAVSQIGASNPGLSGCDVLIQALLEI Consensus VSA.:.IL:SSS:GQ:NYG:::Q.:Q: Ab-Sp-1PCR VSALVHILGSSSVGQINYGASAQYAQM Spidroin 1 VSALIQILGSSSIGQVNYGSAGQATQI Spidroin 2 VSACVTILSSSSIGQVNYGAASQFAQV