Adhesives from Renewable Resources - American Chemical Society

Chapter 32 Development of a Microbial System for Production of Mussel Adhesive Protein Downloaded by UNIV OF SYDNEY on May 29, 2013 | http://pubs.acs.org Publication Date: December 31, 1989 | doi: 10.1021/bk-1989-0385.ch032

R o b e r t L . S t r a u s b e r g , D a v i d M. A n d e r s o n , David Filpula, Malcolm Finkelman, Rebecca

Link,

R u s s e l l M c C a n d l i s s , Steve A. Orndorff, Susan L . S t r a u s b e r g , and T e n a W e i Genex Corporation 16020 Industrial Drive Gaithersburg, M D 20877

The polyphenolic adhesive protein of the mussel Mytilus edulis is an unusual protein composed mainly of repetitive decapeptide and hexapeptide sequences. In the mussel, the protein is first produced in a precursor form and is converted to an adhesive by post-translational modification. To develop an efficient renewable resource for production of the polyphenolic protein, we have used genetic engineering technology. cDNA sequences encoding portions of the polyphenolic protein were identified and expressed in the yeast Saccharomyces cerevisiae. M a r i n e m o l l u s c s reside i n t u r b u l e n t a q u a t i c e n v i r o n m e n t s i n w h i c h s u r v i v a l d e pends u p o n adherence t o a w i d e variety o f surfaces ( i ) . I n response t o such c h a l l e n g i n g c o n d i t i o n s , mussels a n d other molluscs p r o d u c e a byssus for s t r o n g , water-resistant adhesion (1-6). T o t h e biotechnologist, t h e adhesion m e c h a n i s m developed b y these bivalves is o f great interest because o f the need t o f o r m u late adhesives t h a t c a n be a p p l i e d a n d cured i n aqueous e n v i r o n m e n t s a n d t h a t m i g h t b e b i o c o m p a t i b l e . A l t h o u g h the c o m m e r c i a l p o t e n t i a l o f t h i s a n d s i m i l a r adhesive m a t e r i a l s has been well k n o w n for m a n y years, i t s p r o d u c t i o n is quite l i m i t e d i n t h e n a t u r a l host, thereby r e s t r i c t i n g use for m a n y p o t e n t i a l a p p l i c a t i o n s (2). F o r e x a m p l e , t h e mussel adhesive c a n be e x t r a c t e d f r o m t h e p h e n o l g l a n d , b u t o n l y a t quantities o f about 10 m g per mussel ( 7 ) . O u r research has focused o n t h e efficient p r o d u c t i o n o f derivatives a n d analogs o f the m o l l u s c a n adhesives t h r o u g h r e c o m b i n a n t D N A technology. T h i s chapter discusses t h e development o f yeast s t r a i n s for p r o d u c t i o n o f mussel a d 0097-6156/89/0385-0453$06.00/0 * 1989 American Chemical Society

In Adhesives from Renewable Resources; Hemingway, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Downloaded by UNIV OF SYDNEY on May 29, 2013 | http://pubs.acs.org Publication Date: December 31, 1989 | doi: 10.1021/bk-1989-0385.ch032

hesive a n d describes the i m p o r t a n t i m p a c t genetic engineering w i l l have o n the development of a p o t e n t i a l new class of adhesives. W a i t e a n d his colleagues have e x a m i n e d the process of adhesion i n the mussel Mytilus edulis (1,2,7Ί0). T h r o u g h a c o m b i n a t i o n of b i o c h e m i c a l a n d u l t r a s t r u c t u r a l studies, W a i t e has d e t e r m i n e d t h a t byssal adhesion is p r i m a r i l y c o n t r o l l e d by a p o l y p h e n o l i c p r o t e i n (2). C h a r a c t e r i z a t i o n of t h i s p r o t e i n has been g r e a t l y f a c i l i t a t e d b y the o b s e r v a t i o n t h a t the mussel retains s m a l l q u a n t i t i e s i n a p r e a d hesive, n o n c r o s s l i n k e d f o r m i n the p h e n o l g l a n d (8). A series of b i o c h e m i c a l studies has revealed t h a t the p o l y p h e n o l i c p r o t e i n has a n apparent m o l e c u l a r weight of 130,000 d a l t o n s a n d contains repeated decapeptides a n d hexapeptides w i t h the sequence a l a - l y s - p r o - s e r - t y r - h y p - h y p - t h r - d o p a - l y s a n d a l a - l y s - p r o - t h r t y r - l y s a n d h y d r o x y l a t e d derivatives (7-9). B o t h p o s t - t r a n s l a t i o n a l l y h y d r o x y l a t e d residues (i.e., h y d r o x y p r o l i n e a n d dopa) a n d u n h y d r o x y l a t e d residues (i.e., p r o l i n e a n d tyrosine) o c c u r i n the two peptides at the u n d e r l i n e d p o s i t i o n s . T h e p o s t - t r a n s l a t i o n a l h y d r o x y l a t i o n of the tyrosine residues is u n u s u a l a n d l i k e l y p l a y s a n i m p o r t a n t role i n d e t e r m i n i n g a d h e s i v i t y . It has been suggested t h a t o x i d i z e d derivatives of tyrosine residues ( d o p a a n d quinone) p l a y a c r u c i a l role i n adherence a n d c r o s s l i n k i n g a n d t h a t lysine residues are i n v o l v e d i n the crosslinks (*)· W e have several objectives i n m i n d i n a p p l y i n g r e c o m b i n a n t D N A t e c h n o l ogy t o t h e s t u d y a n d a p p l i c a t i o n of the m o l l u s c a n adhesives. B y c l o n i n g D N A sequences e n c o d i n g the p o l y p h e n o l i c p r o t e i n , we w i l l f u r t h e r elucidate the p r i m a r y a m i n o a c i d s t r u c t u r e of t h i s r e p e t i t i v e p r o t e i n . F o r e x a m p l e , sequence a n a l y s i s of the D N A molecule e n c o d i n g the p o l y p h e n o l i c p r o t e i n w i l l reveal whether the p r o t e i n is c o m p o s e d of t a n d e m p e p t i d e repeats, a n d whether there are other u n i q u e regions t h a t also p l a y a role i n adhesivity. S u c h knowledge w i l l p r o v i d e i m p o r t a n t insight i n t o the character of adhesives developed b y n a t u r e for aqueous e n v i r o n m e n t s . E x p r e s s i o n of these cloned D N A sequences i n a m i c r o o r g a n i s m a n d subsequent p u r i f i c a t i o n w i l l p r o v i d e a n i m p o r t a n t renewable resource for efficient p r o d u c t i o n of adhesive proteins. I n a d d i t i o n , the a d v a n c e d state o f D N A c h e m i s t r y p e r m i t s the r a p i d creation of s y n t h e t i c genes t h a t e n code analogs of n a t u r a l adhesive proteins ( A n d e r s o n , D . M . ; S t r a u s b e r g , S. L . ; F i l p u l a , D . ; S t r a u s b e r g , R . L . ; u n p u b l i s h e d d a t a ) . T h r o u g h the use of s y n t h e t i c D N A technology, m i c r o o r g a n i s m s p r o d u c i n g a f a m i l y of adhesive proteins i n w h i c h m e m b e r s differ i n a m i n o a c i d c o m p o s i t i o n a n d m o l e c u l a r weight c a n be generated. W e a n t i c i p a t e t h a t i n t h i s p r o t e i n f a m i l y some members w i l l be i d e a l l y s u i t e d t o a p a r t i c u l a r c o m m e r c i a l a p p l i c a t i o n , whereas, other members of the f a m i l y be m o r e a p p r o p r i a t e for other a p p l i c a t i o n s . T h i s paper describes p r e l i m i n a r y experiments t h a t suggest t h a t efforts i n t h i s area w i l l be successful a n d result i n the development of a new class o f m e d i c a l l y a n d i n d u s t r i a l l y i m p o r t a n t adhesives.


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m R N A I s o l a t i o n a n d C l o n e B a n k P r e p a r a t i o n . P h e n o l g l a n d s were ext r a c t e d f r o m live mussels a n d h o m o g e n i z e d i n l i q u i d n i t r o g e n . T h e frozen tissue was dissolved i n 4 M q u a n i d i n e t h i o c y a n a t e as the first step i n the R N A i s o l a t i o n procedure (11). m R N A was p u r i f i e d f r o m t o t a l R N A u s i n g o l i g o - d ( T ) cellulose c h r o m a t o g r a p h y (12), a n d c D N A was p r e p a r e d as described b y M c C a n d l i s s et a l . (13). T h e c D N A was f r a c t i o n a t e d o n a sucrose gradient because very l i t t l e h i g h m o l e c u l a r weight c D N A was o b t a i n e d . c D N A molecules greater t h a n 500 base p a i r s were p o o l e d a n d cloned i n t o ÀgtlO (14) u s i n g EcoRl linkers. T h e r e c o m b i n a n t D N A was packaged i n t o bacteriophage λ heads for i n t r o d u c t i o n i n t o E. colt (15). A s a host for t i t r a t i o n a n d p r o p a g a t i o n o f the phage, E. coli s t r a i n B N N 1 0 2 (14) was used. H y b r i d i z a t i o n S c r e e n i n g o f t h e C l o n e B a n k . A clone b a n k o f a p p r o x i m a t e l y 500,000 plaques was developed for screening. T h e clone b a n k was p l a t e d at a d e n s i t y of « 25,000 plaques per 14-cm d i s h a n d r e p l i c a t e d i n d u p l i c a t e o n t o nitrocellulose filters (16) for h y b r i d i z a t i o n screening. C l o n e s c a r r y i n g c D N A e n c o d i n g the p o l y p h e n o l i c p r o t e i n were identified b y h y b r i d i z a t i o n w i t h a n o l i g o n u cleotide p r o b e (5' G C G A A A C C A A G T T A C C C A C C G A C C T A C A A A ) . T h e oligonucleotide was r a d i o a c t i v e l y labeled to a specific a c t i v i t y o f a p p r o x i m a t e l y 10 c p m / m g w i t h λ - [ Ρ ] - Α Τ Ρ and T 4 polynucleotidekinase. T h e radioactive oligonucleotides ( a p p r o x i m a t e l y 3.0 m g ) were a d d e d t o 2 5 0 - m L h y b r i d i z a t i o n s o l u t i o n c o n t a i n i n g 2 0 % f o r m a m i d e , 6 X S S C (17), 5 X D e n h a r d t ' s s o l u t i o n , 50m M p h o s p h a t e buffer ( p H 6.8), 100 m g / m L sonicated d e n a t u r e d s a l m o n s p e r m D N A , a n d 1 0 % d e x t r a n sulfate. T h e filters were h y b r i d i z e d for 14 h o u r s at 30 ° C , t h e n washed five t i m e s briefly w i t h 3 0 0 - m L 6 X S S C at 22 ° C , one t i m e w i t h 3 0 0 - m L I X S S C at 22 ° C , a n d once at 42 ° C for 30 m i n u t e s w i t h 5 0 0 - m L I X S S C . T h e filters were a i r - d r i e d a n d a u t o r a d i o g r a p h e d at -80 ° C w i t h K o d a k X A R X - r a y f i l m for 12 h o u r s . 8

3 2

P l a q u e s g i v i n g signals b y a u t o r a d i o g r a p h y o n the d u p l i c a t e filters were p u rified b y p i c k i n g , d i l u t i n g , p l a t i n g , a n d r e p e a t i n g the h y b r i d i z a t i o n screen de s c r i b e d above. Isolated i n d i v i d u a l plaques g i v i n g r a d i o a c t i v e signals were p i c k e d a n d g r o w n as p l a t e lysates for D N A p r e p a r a t i o n (17). D N A S e q u e n c e A n a l y s i s . D N A f r o m A g t l O clone 14-1 was digested w i t h r e s t r i c t i o n endonuclease EcoRl a n d cloned i n t o M 1 3 m p l l (18). T h e b a c t e r i o phage M 1 3 derivatives were constructed u s i n g m e t h o d s described b y M e s s i n g (19). B o t h o r i e n t a t i o n s of the inserts were represented i n i n d e p e n d e n t clones t h a t were sequenced b y the d i d e o x y m e t h o d (20). Y e a s t G e n e t i c s . T h e yeast s t r a i n used i n these studies was Y G X D 8 ( M A T a /ew2-3 /ew2-112). Y e a s t cells were t r a n s f o r m e d by the spheroplast m e t h o d of H i n n e n et a l . (21). T h e t r a n s f o r m e d cells were m a i n t a i n e d i n Y N B m e d i u m ( 0 . 7 % yeast n i t r o g e n base) s u p p l e m e n t e d w i t h 5 % glucose. F o r i n d u c t i o n of p o l y p h e n o l i c p r o t e i n synthesis, the cells were c u l t u r e d i n Y P m e d i u m ( 1 % yeast e x t r a c t , 1% bacto-peptone) s u p p l e m e n t e d w i t h 4 % glucose a n d 2 % galactose.


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Results and Discussion


Isolation a n d Characterization of c D N A Clones E n c o d i n g the Polyp h e n o l i c P r o t e i n . C h a r a c t e r i z a t i o n of the p r i m a r y a m i n o a c i d sequence of the mussel adhesive p r o t e i n has been h i n d e r e d b y the large size o f the p r o t e i n a n d the repetitiveness of the a m i n o acids. I n s u c h cases, the p r a c t i c a l ( a n d p e r h a p s o n l y ) a p p r o a c h for d e t e r m i n i n g the complete a m i n o a c i d sequence is to clone D N A sequences e n c o d i n g the p r o t e i n a n d to deduce the a m i n o a c i d sequence f r o m the genetic code c a r r i e d b y t h a t D N A . T o a c c o m p l i s h t h i s , we o b t a i n e d m R N A f r o m mussels a n d synthesized c D N A in vitro. T o screen for clones c a r r y i n g c D N A e n c o d i n g the p o l y p h e n o l i c p r o t e i n , h y b r i d i z a t i o n w i t h a n oligonucleotide p r o b e (5' G C G A A A C C A A G T T A C C C A C C G A C C T A C A A A ) was p e r f o r m e d . T h e p r o b e was designed based o n the a m i n o a c i d sequence of the d e c a p e p t i d e identified b y W a i t e (9). Because o f the degeneracy o f the genetic code, i t was not k n o w n h o w s i m i l a r t h i s oligonucleotide w o u l d be t o sequences e n c o d i n g the d e c a p e p t i d e i n the mussel. Therefore, n o n stringent h y b r i d i z a t i o n c o n d i t i o n s a n d d u p l i c a t e filters were u t i l i z e d to i d e n t i f y p o t e n t i a l p o s i t i v e clones. A p p r o x i m a t e l y 20 clones p o t e n t i a l l y c a r r y i n g c o d i n g sequence for the p o l y p h e n o l i c p r o t e i n were identified. T h e first clone t o be f u l l y characterized by D N A sequencing was clone 14-1 ( F i g u r e 1). T h i s clone appears to c a r r y c o d i n g i n f o r m a t i o n for the c a r b o x y l term i n u s of the p o l y p h e n o l i c p r o t e i n . It contains the t y p i c a l e u c a r y o t i c p o l y a d e n y l a t i o n s i g n a l ( A A T A A A ) , a p o l y A t a i l , a n d a 216 base 3' n o n t r a n s l a t e d r e g i o n . T h e c o d i n g sequence of clone 14-1 provides interesting insight i n t o the org a n i z a t i o n of repeat u n i t s i n the p o l y p h e n o l i c p r o t e i n . T h e entire p o l y p e p t i d e sequence encoded b y t h i s clone consists of related peptides o r g a n i z e d as t a n d e m repeats ( F i g u r e 2). C o d i n g sequences for the d e c a p e p t i d e a l a - l y s - p r o - s e r t y r - p r o - p r o - t h r - t y r - l y s , identified b y W a i t e as a n i m p o r t a n t c o m p o n e n t o f the p o l y p h e n o l i c p r o t e i n , are present four t i m e s i n the clone 14-1. However, the sequence c o m p l e x i t y of the repeat u n i t s is m u c h greater t h a n e x p e c t e d , since o u t o f 19 decapeptides e n c o d e d b y clone 14-1, 14 different a m i n o a c i d sequences are observed. A n e x a m i n a t i o n of these sequences provides useful insight i n t o key a m i n o a c i d o r g a n i z a t i o n i n the mussel adhesive. A m i n o acids at p o s i t i o n s 2 (lys), 5 ( t y r ) , 6 ( p r o ) , a n d 9 (tyr) are i d e n t i c a l i n a l l decapeptides, a n d o n l y a single d e c a p e p t i d e w i t h o u t lysine at p o s i t i o n 10 is identified. T h e h i g h level of conservation of tyrosine a n d lysine residues suggests a n i m p o r t a n t role for these a m i n o acids a n d their p o s t - t r a n s l a t i o n a l derivatives i n b o t h adhesive a n d cohesive processes. A s m e n t i o n e d earlier, W a i t e has p r o p o s e d t h a t l y s i n e a n d quinone residues are i n v o l v e d i n p r o t e i n c r o s s l i n k i n g . S u b s t i t u t i o n s at other p o s i t i o n s generally result i n the presence o f serine, t h r e o n i n e , p r o l i n e (perhaps h y d r o x y p r o l i n e ) , a l a n i n e , a n d isoleucine residues, w i t h a n e m phasis o n p o l a r residues t h a t can interact w i t h m o s t b i o l o g i c a l surfaces. T h e r e s u l t i n g p r o t e i n is r i c h i n the s i x a m i n o acids: tyrosine, l y s i n e , a l a n i n e , serine, t h r e o n i n e a n d p r o l i n e . P r e l i m i n a r y c h a r a c t e r i z a t i o n of c D N A clones e n c o d i n g


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5 10 15 Lys Pro Lys Pro Ser Tyr Pro Pro Ser Tyr Lys Pro Lys Thr Thr Tyr Pro GAA TTC CAT AAA CCA AAA CCA AGT TAT CCA CCA TCT TAT AAA CCT AAA ACA ACT TAT CCT


EcoRl 20 25 30 35 Pro Thr Tyr Lys Pro Lys I l e Ser Tyr Pro Pro Thr Tyr Lys A l a Lys Pro Ser T y r Pro CCA ACT TAT AAA CCT AAG ATA AGT TAT CCT CCA ACT TAT AAA GCA AAA CCA AGT TAT CCA 40 45 50 55 A l a Thr Tyr Lys A l a Lys Pro Ser Tyr Pro Pro Thr T y r Lys A l a Lys Pro Ser T y r Pro GCA ACT TAT AAA GCA AAA CCA AGT TAT CCT CCA ACT TAT AAA GCA AAA CCA AGT TAT CCT 60 65 70 75 Pro Thr Tyr Lys A l a Lys Pro Ser Tyr Pro Pro Thr T y r Lys A l a Lys Pro Thr T y r Lys CCA ACT TAT AAA GCA AAA CCA AGT TAT CCT CCA ACT TAT AAA GCA AAG CCA ACT TAT AAA 80 85 90 95 A l a Lys Pro Thr T y r Pro Pro Thr T y r Lys A l a Lys Pro Ser T y r Pro Pro Thr T y r Lys GCA AAG CCA ACT TAT CCT CCA ACT TAT AAA GCA AAA CCA AGT TAT CCT CCA ACA TAT AAA 100 105 110 115 Pro Lys Pro Ser T y r Pro Pro Thr Tyr Lys Ser Lys Ser H e T y r Pro Ser Ser T y r Lys CCA AAG CCA AGT TAT CCT CCA ACT TAT AAA TCC AAG TCA ATA TAT CCC TCT TCA TAC AAA 120 125 130 135 Pro Lys Lys Thr T y r Pro Pro Thr Tyr Lys Pro Lys Leu Thr T y r Pro Pro Thr T y r Lys CCT AAG AAA ACT TAT CCC CCC ACA TAT AAA CCT AAA CTA ACC TAT CCT CCA ACA TAT AAA 140 145 150 155 Pro Lys Pro Ser Tyr Pro Pro Ser Tyr Lys Pro Lys I l e Thr T y r Pro Ser Thr Tyr Lys CCA AAG CCA AGT TAT CCA CCA TCT TAT AAA CCT AAG ATT ACT TAT CCC TCA ACT TAT AAA 160 165 170 175 Leu Lys Pro Ser T y r Pro Pro Thr Tyr Lys Ser Lys Thr Ser T y r Pro Pro Thr T y r Asn TTG AAG CCA AGT TAT CCT CCA ACA TAC AAA TCT AAA ACA AGT TAC CCT CCT ACA TAT AAC 180 185 190 195 Lys Lys I l e Ser T y r Pro Ser Ser Tyr Lys A l a Lys Thr Ser T y r Pro Pro A l a T y r Lys AAA AAG ATC AGC TAT CCA TCA TCA TAT AAA GCT AAG ACA AGT TAT CCC CCA GCA TAT AAA 200 Pro Thr Asn Arg Tyr *** CCA ACA AAC AGA TAT TAA TCT CAA TAT TAA AAG TAT TAA CTA AAA TAT TCA CAT TAC TGT ACT ACA CAT TTT AAC GTT TGT ATT GAT GAG GAA CAG ATG AAC ATT TGA AAG TAA TAC ATA ATC GGG GTT AAT GAT TTG TTA TAT TCA ATC TTA ATA TGT TTG TGA TTT GTT ATG TTC TTG AAG TAT TGT TTC AAA TAA AGT TTA TTC TTT TCT GGT AAA AAA AAA AAA AAA GGA ATT CC £coRl

F i g u r e 1. D N A sequence a n d t r a n s l a t i o n p r o d u c t o f 14-1 c D N A clone. T h e EcoRl sites at the 5' a n d 3' ends were generated b y oligonucleotide l i n k e r s .



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ADHESIVES F R O M RENEWABLE RESOURCES

larger segments of the p o l y p h e n o l i c p r o t e i n demonstrates t h a t r e p e t i t i v e a m i n o a c i d sequences account for m o s t o f the p r o t e i n m o l e c u l a r weight. I n s e r t i o n of t h e 14-1 c D N A Sequence I n t o a Y e a s t E x p r e s s i o n V e c t o r . B a s e d o n the e x p e c t a t i o n t h a t lower m o l e c u l a r weight derivatives of the p o l y p h e n o l i c p r o t e i n m i g h t show adhesive properties ( a l t h o u g h tensile s t r e n g t h m i g h t be lower t h a n for a f u l l - l e n g t h p r o t e i n ) , a m i c r o b i a l p r o d u c t i o n s y s t e m was developed for t h i s p r o t e i n . Efficient expression of the p o l y p h e n o l i c p r o t e i n i n m i c r o o r g a n i s m s presents a n u n u s u a l challenge because o f t w o c h a r a c t e r i s tics o f the p o l y p h e n o l i c p r o t e i n . F i r s t , because the p r o t e i n is composed o f a n u n u s u a l s p e c t r u m o f a m i n o acids, the m i c r o b i a l cell m i g h t have difficulty i n t r a n s l a t i n g the m R N A for t h i s p r o t e i n . F o r e x a m p l e , i t has been suggested t h a t c o d o n choice i n a heterologous m R N A m a y reduce m i c r o b i a l expression of foreign p r o t e i n s because of l i m i t i n g t R N A molecules i n the cell (22). That effect c o u l d be m o r e p r o n o u n c e d for a r e p e t i t i v e p r o t e i n encoded b y r e l a t i v e l y few codons. Second, because the D N A sequence e n c o d i n g the p r o t e i n is also h i g h l y r e p e t i t i v e , D N A r e c o m b i n a t i o n m i g h t generate u n e q u a l crossover events (23) t h e r e b y r e s u l t i n g i n p r o d u c t i o n o f a n a r r a y o f p o l y p h e n o l i c derivatives a n d r e d u c i n g the r e p r o d u c i b i l i t y a n d q u a l i t y of the p r o d u c t . y

I n spite o f these p o t e n t i a l difficulties, we have been successful i n d e v e l o p i n g a n efficient yeast p r o d u c t i o n s y s t e m for the p o l y p h e n o l i c p r o t e i n . T h e yeast Saccharomyces cerevisiae was chosen for these studies because i t is a safe o r g a n i s m w i t h G R A S s t a t u s ; i t n a t u r a l l y carries stable r e p e t i t i v e D N A sequences e n c o d i n g p r o t e i n p r o d u c t s (24,25), a n d we have r e c e n t l y developed expression vectors capable o f p r o d u c i n g foreign proteins at levels o f u p to 2 0 % of the t o t a l cell p r o t e i n ( S t r a u s b e r g , R . L . ; S t r a u s b e r g , S. L . ; u n p u b l i s h e d d a t a ) . T h i s yeast has p r e v i o u s l y been used for p r o d u c t i o n of a v a r i e t y o f p h a r m a c e u t i c a l p r o d u c t s (26-30). T o i n t r o d u c e the p o l y p h e n o l i c p r o t e i n c o d i n g sequence i n t o yeast, the D N A sequence was transferred f r o m a n M 1 3 vector used for D N A sequence a n a l y s i s i n t o a yeast-E. coli s h u t t l e vector, Y p G X 2 8 5 ( F i g u r e 3). T h i s vector carries r e p l i c a t i o n o r i g i n s a n d selectable m a r k e r s b o t h for E. coli a n d S. cerevisiae, thereby p e r m i t t i n g genetic c o n s t r u c t i o n s t o be c o m p l e t e d u s i n g E. coli cells, w h i c h simplifies the m a n i p u l a t i o n s . F o r p l a s m i d r e p l i c a t i o n a n d m a i n t e n a n c e i n yeast, the p l a s m i d carries a r e p l i c a t i o n o r i g i n f r o m the n a t u r a l yeast 2 - m i c r o n p l a s m i d a n d the L E U 2 - D gene as a selectable m a r k e r (31). B y g r o w t h i n m e d i a l a c k i n g exogenous leucine, t h i s p l a s m i d can be easily m a i n t a i n e d i n yeast cells c a r r y i n g m u t a t i o n s i n the c h r o m o s o m a l L E U 2 gene. F o l l o w i n g t r a n s f o r m a t i o n , the L E U 2 gene p r o d u c t p r o v i d e d b y the p l a s m i d - b o r n e gene allows the cells t o grow i n m e d i a l a c k i n g leucine. F o r expression o f the p o l y p h e n o l i c p r o t e i n , the c o d i n g sequence is inserted i n t o a n expression cassette composed of a p r o m o t e r , t r a n s l a t i o n i n i t i a t i o n sequence, s i g n a l sequence, a n d t r a n s c r i p t i o n t e r m i n a t o r . T h e p r o m o t e r sequence i n Y p G X 2 8 5 is derived f r o m t w o yeast genes, G A L 1 (32) a n d a l p h a factor ( M F al) (25). T h i s h y b r i d p r o m o t e r provides for efficient, r e g u l a t e d , t r a n s c r i p t i o n of



32.

S T R A U S B E R G

Microbial

E T A L

Pro Pro Pro Ala Ala Ala Ala Ala Ala Ala Pro Ser Pro Pro Pro Pro Leu Ser Lys Ala

Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

Pro Thr Ile Pro Pro Pro Pro Pro Pro Pro Pro Ser Lys Leu Pro Ile Pro Thr Ile Thr

System for Mussel Adhesive

Tyr Tyr Tyr Tyr Tyr Tyr Tyr

Pro Pro Pro Pro Pro Pro Pro

Pro Pro Pro Ala Pro Pro Pro

Thr Tyr Ser Tyr Ser Tyr H e Tyr Thr Tyr Thr Tyr Ser Tyr Thr Tyr Ser Tyr Ser Tyr Ser Tyr Ser Tyr

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro

Pro Pro Pro Ser Pro Pro Pro Ser Pro Pro Ser Pro

Ser Thr Ser Ser Ser Ser Ser

Ser Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ser Thr Thr Ser Thr Thr Thr Ser Ala

Protein

Tyr Lys T y r Lys Tyr Lys T y r Lys T y r Lys T y r Lys T y r Lys T y r Lys Tyr Lys T y r Lys Tyr Lys T y r Lys T y r Lys T y r Lys T y r Lys Tyr Lys T y r Lys T y r Asn Tyr Lys T y r Lys

F i g u r e 2. R e p e a t sequences encoded b y the 14-1 c D N A clone.


459

460

ADHESIVES F R O M R E N E W A B L E RESOURCES


D N A sequences p l a c e d under i t s c o n t r o l . E x p r e s s i o n of foreign genes is t i g h t l y c o n t r o l l e d s i m p l y b y a d d i n g galactose t o the c u l t u r e m e d i a , w h i c h induces ex pression f r o m t h i s p r o m o t e r . S u c h r e g u l a t e d expression of foreign gene p r o d u c t s is often desirable or essential because c o n s t i t u t i v e expression o f the foreign gene p r o d u c t c a n l i m i t c e l l u l a r g r o w t h , r e s u l t i n g i n selective pressure for n o n p r o d u c i n g cells i n the p o p u l a t i o n . T h e p l a s m i d carries a P H 0 5 (33) s i g n a l - c o d i n g sequence i m m e d i a t e l y 3' t o the p r o m o t e r sequence. N o r m a l l y , a s i g n a l - c o d i n g sequence is present i n a n ex pression vector t o direct secretion o f the foreign p r o t e i n f r o m the cell. However, for expression of the p o l y p h e n o l i c p r o t e i n , the P H 0 5 s i g n a l sequence is present m a i n l y t o p r o v i d e for efficient t r a n s l a t i o n i n i t i a t i o n of the foreign gene p r o d u c t . A m e t h i o n i n e residue is p o s i t i o n e d between the P H 0 5 s i g n a l a n d p o l y p h e n o l i c p r o t e i n t o p r o v i d e a cyanogen b r o m i d e r e c o g n i t i o n site for in vitro e x c i s i o n of the s i g n a l sequence. T h e Y p G X 2 8 5 vector also carries a t r a n s c r i p t i o n t e r m i n a t i o n sequence derived f r o m a yeast g l y c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase ( G A P D H ) (34) gene. M i c r o b i a l E x p r e s s i o n of t h e 14-1 c D N A E n c o d e d P o l y p h e n o l i c P r o t e i n . F o l l o w i n g t r a n s f o r m a t i o n o f a h a p l o i d yeast s t r a i n w i t h the Y p G X 2 8 5 expression vector, i n i t i a l studies were p e r f o r m e d at the shake-flask level t o de t e r m i n e w h e t h e r p o l y p h e n o l i c p r o t e i n was p r o d u c e d b y the yeast cells u p o n galactose i n d u c t i o n . T h o s e studies d e m o n s t r a t e d t h a t the cells were p r o d u c i n g the p o l y p h e n o l i c p r o t e i n d e r i v a t i v e a n d t h a t the p r o d u c t was homogeneous a n d o f the e x p e c t e d m o l e c u l a r weight. T h e r e f o r e , f e r m e n t a t i o n studies were c o n d u c t e d , first at the 2-liter scale a n d subsequently i n 10-liter a n d 250-liter fermentors t o determine efficiency o f p r o d u c t i o n a n d s t a b i l i t y o f the r e c o m b i n a n t s t r a i n . A n e x a m p l e o f the results of these studies is s h o w n i n F i g u r e 4. I n t h i s e x p e r i m e n t , a b a t c h f e r m e n t a t i o n was p e r f o r m e d at the 2-liter scale. T h e cells were fermented i n Y P m e d i u m c o n t a i n i n g 4 % glucose a n d 2 % galactose as the c a r b o n sources, a n d the i n s o l uble c e l l u l a r p r o t e i n was e x a m i n e d by S D S p o l y a c r y l a m i d e gel electrophoresis (35). E a r l y i n the f e r m e n t a t i o n , as glucose was b e i n g u t i l i z e d , expression o f the p o l y p h e n o l i c p r o t e i n was not observed ( F i g u r e 4, lane A ) . A s the glucose was depleted a n d galactose m e t a b o l i s m c o m m e n c e d ( F i g u r e 4, lanes Β a n d C ) , p r o d u c t i o n o f the p o l y p h e n o l i c p r o t e i n began a n d reached a steady-state level o f a b o u t 5 % o f the t o t a l cell p r o t e i n ( F i g u r e 4, lanes D - F ) . P h y s i o l o g i c a l studies o f the r e c o m b i n a n t yeast have s h o w n t h a t m a i n t e n a n c e o f the cells i n m e d i a c o n t a i n i n g o n l y glucose as the c a r b o n source results i n g o o d genetic s t a b i l i t y a n d c e l l u l a r g r o w t h rates c o m p a r a b l e t o those of the n o n r e c o m b i n a n t host s t r a i n . I n a d d i t i o n , the p l a s m i d - b o r n e p o l y p h e n o l i c c o d i n g sequences undergo very l i t t l e genetic r e c o m b i n a t i o n . T h e l a t t e r finding was u n e x p e c t e d because a p r i o r i we a n t i c i p a t e d t h a t a very r e p e t i t i v e D N A sequence c a r r i e d b y a p l a s m i d r e p l i c a t i n g at h i g h copy n u m b e r i n the cell w o u l d be a g o o d s u b s t r a t e for genetic r e c o m b i n a t i o n . However, t h a t has not been the case



32.

S T R A U S B E R G E T A L

Microbial


461

F i g u r e 4. R e g u l a t e d p r o d u c t i o n of p o l y p h e n o l i c p r o t e i n i n yeast. T h e arrow i n d i c a t e s the p o l y p h e n o l i c p r o t e i n . P u r i f i e d yeast-derived p o l y p h e n o l i c p r o t e i n , t r e a t e d in viiro w i t h cyanogen b r o m i d e , is present i n lane G .


462


a n d suggests t h a t yeast m a y be a very g o o d p r o d u c t i o n o r g a n i s m for p r o t e i n s encoded b y r e p e t i t i v e D N A sequences.


A f t e r f e r m e n t a t i o n , the yeast cells were harvested a n d b r o k e n m e c h a n i c a l l y i n a b e a d m i l l , a n d differential c e n t r i f u g a t i o n was used t o p a r t i t i o n the c e l l u l a r c o m p o n e n t s i n t o water-soluble a n d i n s o l u b l e f r a c t i o n s . S D S - p o l y a c r y l a m i d e gel electrophoresis i n c o n j u n c t i o n w i t h W e s t e r n b l o t a n a l y s i s (36) revealed t h a t the p o l y p h e n o l i c p r o t e i n aggregated w i t h the i n s o l u b l e c e l l u l a r p r o t e i n . F o r the p r o d u c t i o n of m a n y r e c o m b i n a n t p r o t e i n s , c e l l u l a r i n s o l u b i l i t y is a m a j o r p r o b l e m because i t is often difficult to achieve b i o l o g i c a l a c t i v i t y s t a r t i n g w i t h such p r o d u c t s (37). However, because the p o l y p h e n o l i c p r o t e i n p r o d u c e d in vivo is a preadhesive, in vitro a c t i v a t i o n t o the adhesive f o r m is r e q u i r e d . Therefore, in vivo i n s o l u b i l i t y m a y a c t u a l l y be desirable i n the case o f the p o l y p h e n o l i c p r o t e i n because t h i s c o u l d result i n increased resistance t o yeast proteases a n d better p r o d u c t u n i f o r m i t y a n d q u a n t i t y . P u r i f i c a t i o n o f t h e P o l y p h e n o l i c P r o t e i n f r o m Y e a s t . Unlike most insoluble yeast p r o t e i n s , the p o l y p h e n o l i c p r o t e i n is h i g h l y basic. T h i s suggested t h a t a n efficient p u r i f i c a t i o n o f the p o l y p h e n o l i c p r o t e i n c o u l d b e achieved b y a c i d e x t r a c t i o n o f the t o t a l i n s o l u b l e p r o t e i n . A f t e r the cells were b r o k e n m e c h a n i c a l l y , c e l l u l a r m a t e r i a l s were segregated b y c e n t r i f u g a t i o n i n t o water-soluble a n d i n s o l u b l e f r a c t i o n s . T h e i n s o l u b l e m a t e r i a l was e x t r a c t e d i n t o 7 0 % f o r m i c a c i d , r e s u l t i n g i n s o l u b i l i z a t i o n of the p o l y p h e n o l i c p r o t e i n . A f t e r r e m o v a l of the a c i d b y r o t a r y e v a p o r a t i o n a n d several washes w i t h w a t e r , the p o l y p h e n o l i c p r o t e i n was p r e c i p i t a t e d w i t h 1 0 % s o d i u m chloride i n a d i l u t e a c i d i c s o l u t i o n . T h e p o l y p h e n o l i c p r o t e i n is a b o u t 6 0 % pure o n a weight basis at t h i s stage ( T . W e i , u n p u b l i s h e d d a t a ) . F o l l o w i n g r e s o l u b i l i z a t i o n i n 6 M g u a n i d i n e h y d r o c h l o r i d e a n d 5 % 2 - m e r c a p t o e t h a n o l , the p a r t i a l l y p u r i f i e d p r o t e i n s were c h r o m a t o g r a p h e d o n a S e p h a c r y l S-300 c o l u m n . F r a c t i o n s c o n t a i n i n g the h i g h l y p u r i f i e d p o l y p h e n o l i c p r o t e i n were p o o l e d , a d j u s t e d to p H 4.0 w i t h acetic a c i d , d i a l y z e d against 0 . 1 % acetic a c i d , a n d t h e n l y o p h i l i z e d . T h e recovered p o l y p h e n o l i c p r o t e i n is at least 9 0 % p u r e o n a weight basis. If desired, the yeast P H 0 5 s i g n a l sequence c a n be excised f r o m the p o l y p h e n o l i c p r o t e i n b y cyanogen b r o m i d e cleavage. Conversion of the Microbially P r o d u c e d Preadhesive to an Adhesive P r o t e i n . T h e p o l y p h e n o l i c p r o t e i n p u r i f i e d f r o m yeast adheres t o a w i d e v a r i e t y o f surfaces i n c l u d i n g glass a n d p l a s t i c . T h e adherence p r o b a b l y results f r o m the presence o f m a n y p o l a r residues capable o f h y d r o g e n b o n d i n g a n d l y sine residues t h a t c a n f o r m i o n i c i n t e r a c t i o n s . However, t h i s p r o t e i n does not generate water-resistant b o n d s t o surfaces nor does i t have cohesive s t r e n g t h . F o r those purposes, i t is necessary t o convert at least a p o r t i o n o f the t y r o s i n e residues t o d o p a a n d p e r m i t crosslink f o r m a t i o n t o o c c u r after surface adhesion is achieved. T h a t i s , i t is necessary t o m i m i c the n a t u r a l mussel process i n w h i c h the d o p a f o r m o f the p o l y p h e n o l i c p r o t e i n is a p p l i e d a n d t h e n r a p i d l y



32.

S T R A U S B E R G E T A L

Microbial


463

c r o s s l i n k e d . Therefore, i t i s i m p o r t a n t t o d e m o n s t r a t e t h a t these p r o t e i n s c a n be h y d r o x y l a t e d in vitro. M u s h r o o m tyrosinase has p r e v i o u s l y been used t o convert t y r o s i n e residues i n c h e m i c a l l y synthesized p o l y p h e n o l i c decapeptides t o d o p a residues (38). T h i s e n z y m e also c a n convert d o p a residues t o quinones, b u t t h e e n z y m a t i c p r o d u c t can b e m a i n t a i n e d i n the d o p a f o r m i f r e d u c i n g c o n d i t i o n s are u t i l i z e d . U s i n g m u s h r o o m tyrosinase, we have converted a t least 5 0 % o f t h e t y r o s i n e residues to d o p a a n d have evidence f o r q u i n o n e - l y s i n e crosslinks i n a n o x i d i z i n g e n v i r o n m e n t ( T . W e i a n d R . L i n k , u n p u b l i s h e d d a t a ) . W h e n these c o n d i t i o n s are carefully c o n t r o l l e d , we have observed adhesive properties for t h e r e c o m b i n a n t p o l y p h e n o l i c p r o t e i n . W e are c u r r e n t l y s t u d y i n g t h e p a r a m e t e r s t h a t c a n i n crease a d h e s i v i t y a n d m o i s t u r e resistance t h r o u g h better surface i n t e r a c t i o n s a n d m o r e extensive c r o s s l i n k i n g . Conclusions Future Research to Develop Genetically Engineered Molluscan A d h e s i v e s . T h e p r e l i m i n a r y tests o f the r e c o m b i n a n t p o l y p h e n o l i c p r o t e i n suggest t h a t , w h e n h y d r o x y l a t e d , t h i s t y p e o f p r o t e i n w i l l have t h e expected properties of s t r o n g , m o i s t u r e - r e s i s t a n t adhesion. S t u d i e s c u r r e n t l y u n d e r w a y w i l l reveal w h e t h e r t h i s p r o t e i n w i l l b e n o n t o x i c a n d n o n i m m u n o g e n i c f o r in vivo m e d i c a l a n d d e n t a l uses. A l t h o u g h c h a r a c t e r i z a t i o n o f t h e m i c r o b i a l l y p r o d u c e d mussel p o l y p h e n o l i c p r o t e i n is i n i t s early stages, i t i s l i k e l y t h a t these studies w i l l p r o v i d e i m p o r t a n t i n s i g h t i n t o n a t u r e ' s m e c h a n i s m s for s o l v i n g t h e p r o b l e m o f l o n g - l a s t i n g m o i s t u r e - r e s i s t a n t adhesion. I t w i l l b e i n t e r e s t i n g t o c o m p a r e the m e c h a n i s m devised b y t h e mussel w i t h t h a t o f other invertebrates s u c h as the b a r n a c l e . O u r g o a l is t o p r o d u c e these generally u n a v a i l a b l e adhesives i n r e c o m b i n a n t m i c r o b i a l systems, thereby p r o v i d i n g a n e c o n o m i c a l , renewable, c o m m e r c i a l p r o d u c t i o n source. A cknowledgment s T h e a u t h o r s t h a n k D r . J u d i t h H a u t a l a for h e l p f u l discussions a n d L o i s M o s e r D i n t e r m a n , S t e p h e n P u l f o r d , a n d L i s a R a y m o n d f o r excellent t e c h n i c a l assistance. W e also w i s h t o t h a n k L o r r a i n e L o r e n z for t y p i n g t h e m a n u s c r i p t . Literature Cited 1. Waite, J. H. Biochem. Rev. Cambridge Philos. Soc. 1983,58, 209. 2. Waite, J. H. Int. J. Adhesion and Adhesives 1987, 7(1), 9. 3. Jackson, S. F.; Kelly, F.C.;North, A.C.T.; Randall, J. T.; Seeds, W. E.; Watson, M.; Wilkinson, G. R. In Nature and Structure of Collagen; Randall, J. T.; Jackson, S. F., Eds.; Butterworths: London, 1985; p. 153. 4. Price, H. A. J. Zool. 1981, 194, 245.


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5. Pujol, J. P.; Rolland, M.; Lasry, S.; Vinet, S. Comp. Biochem. Biophys. 1970, 34, 193. 6. Smeathers, J. E.; Vincent, J.F.V. J. Molluscan Stud. 1979, 45, 219. 7. Waite, J. H. J. Biol Chem. 1983, 258, 2911. 8. Waite, J. H.; Tanzer, M. L. Science 1981, 212, 1038. 9. Waite, J. H.; Houseley, T.; Tanzer, M. L. Biochemistry 1985, 24, 5010. 10. Benedict, C. V.; Waite, J. H. J. Morphology 1986, 189, 261.


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