enhancement of accessibility of cellulose by expansins

(12) United States Patent Cosgrove

US006326470B1

(10) Patent N0.: (45) Date of Patent:

US 6,326,470 B1 Dec. 4, 2001

(54) ENHANCEMENT OF ACCESSIBILITY OF CELLULOSE BY EXPANSINS

(75) Inventor: Daniel J. Cosgrove, Pennsylvania Furnace, PA (US)

(73) Assignee: The Penn State Research Foundation, University Park, PA (US)

( * ) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days.

(21) Appl. No.: 09/429,675

(22) Filed: Oct. 28, 1999

Related US. Application Data

63 Continuation-in- art of a lication No. 08/834,327, ?led on P PP A r. 15 1997 now Pat. No. 5 990 283. P 7 7 7 7

60 Provisional a lication No. 60/105,906, ?led on Oct. 28, PP 1998.

nt. . ........................... .. ; ; 51 I C] 7 C07K 1/00 C07K 14/00 C07K 16/00; C07K 17/00

(52) US. Cl. ........................ .. 530/370; 530/372; 530/375; 530/376; 530/377; 530/378; 530/379; 435/183;

435/195; 435/209 (58) Field of Search ................................... .. 530/370, 372,

530/375, 376, 377—379; 435/183, 195, 209

(56) References Cited

U.S. PATENT DOCUMENTS

3,844,890 10/1974 Horikoshi et al. ................... .. 195/62

3,966,543 6/1976 Cayle et al. .. 162/158 4,004,976 1/1977 Isaac ............... .. 565/598

5,175,275 12/1992 Dobashi et al. 536/56 5,959,082 * 9/1999 Cosgrove et al. ................. .. 530/370

OTHER PUBLICATIONS

McQueen—Mason et al. Proc. Natl. Acad. Sci. USA, vol. 91, pp. 6574—6578, Jul. 1994.* Fry, Stephen C., Physiologia Plantarium 75:532—536, 1989. Patel, Praful, in “Biotechnology Applications and

Research”, eds. Cheremisinoff, RN. and Ouellette, R.P. pp. 534—562, Technomic Publishing Company, Inc., Lancaster, PA, 1985.

(List continued on next page.)

Primary Examiner—DWayne C. Jones Assistant Examiner—C. Delaceroix-Muirheid

(74) Attorney, Agent, or Firm—Zarley, McKee, Thomte, Voorhees & Sease

(57) ABSTRACT

Plant cell expansion is regulated by Wall relaxation and yielding, Which is thought to be catalyzed by elusive “Wall loosening” enZymes. By employing a reconstitution approach, We initially found that a crude protein extract from the cell Walls of groWing cucumber seedlings possessed the ability to induce the extension of isolated cell Walls. This activity Was restricted to the groWing region of the stem and could induce the extension of isolated cell Walls from various dicots and monocots, but Was less effective on grass coleoptile Walls. Sequential HPLC fractionation of the active Wall extract revealed tWo proteins With molecular masses of 29 and 30 kD, as measured by SDS-PAGE, associated With such activity. Each protein, by itself, could induce Wall extension Without detectable hydrolytic break doWn of the Wall. We proposed the name “expansins” for this class of proteins. Expansins have been isolated from various plant sources including oat, cucumber, broccoli, celery, tomato, cotton, cabbage, and corn, and also from snail and its feces. These proteins Weaken the intermolecular bonds betWeen plant Wall polysaccharides. They decrease the mechanical strength of commercial products made from polysaccharides, such as paper, and therefore present a novel approach in developing neW technologies in industries Which make use of such polysaccharides, such as in the paper industry, in the applications of polysaccharide gums and related products. These proteins moreover present a novel approach in the control of plant groWth.

15 Claims, 33 Drawing Sheets

US 6,326,470 B1

OTHER PUBLICATIONS

TaiZ, L., Ann. Rev. Plant Physiol. 35:585—657, 1984. Scopes, R.K., Chapter 3, Protein Puri?cation, Principles and Practice, 2nd Ed., pp. 41—54, Springer—Verlag, New York, 1987. Deutscher, M.P., Academic Press 1990, Guide to Protein Puri?cation, pp. 174—193. Fry, Stephen C., Current Biology 4(9):815—817, 1994. Cleland, R.E., Planta 170:379—385, 1987. Nishitani, K.and Torninaga R., J. Biol. Chem. 267(29):21058—21064, 1992. TaiZ, L., Proc. Natl. Acad. Sci. USA 91:7387—7389, Aug. 1994.

Shcherban, T.Y. et al., Proc. Natl. Acad. Sci. USA 92:9245—9249, Sep. 1995. Bayer, EA. et a1., Current Opinion in Structural—Biology 8:548—557, 1998. Cosgrove, D]. et al., Proc. Natl. Acad. Sci. USA 94:6559—6564, 1997. Li, Z—C et a1., Planta 191:349—356, 1993. McQueen—Mason, S. and Cosgrove, DJ, Proc. Natl. Sci. USA 911657416578, 1994. McQueen—Mason, S. and Cosgrove, DJ, Plant Physiol. 107:87—100, 1995. McQueen—Mason, S. et a1., Planta 190:327—331, 1993.

* cited by eXarniner

U.S. Patent Dec. 4, 2001 Sheet 1 0f 33 US 6,326,470 B1

Wall: Native

pH 6.8 to 4.5

i Boiled /

+ Apical Wall Proteins

l + Soluble Proteins Boned

+ Basal Wall Proteins Boiled

/l/'/ + Apical Wall Proteins

200 pm l—-—-|

Change in Length

Basal —— (Native)

1 I l l l I 0.0 0.5 1.0 1.5 2.0 2.5

Fig.1A


Tomato

E :1.

8 I + Cucumber Pea N Wall Protein

Radish 5 . g Llly a» ._l

.E Union 0 Cl C ea .1‘:

/Barley I I I F

0 0 0 5 1.0 1 5 2.0

Fig.1B


AU (280 nm) C: .A ‘w b

1

I .8; Activity (0/. m")

0-0 -- ...___.______._..--v'

I I T I T T I I I I I I I [j l I TTT T I I I I Ii

0 10 20 30 40 50

Retention Tlme (min)

Fig.2A

0.025"

0.020‘

0.015-1

AU (280 nm) omo-J

Activity (% hr") 0.005'1

0.000‘“

?l'ITrlTIlIlIIlp?lIIl'II 0 10 20 30 40

Retention Tame (min)

Fig.2B


Mm} 1500.0 -—l>

am:

49.5 _"

32.5 —l'

215 “b

Fig.5


pH 6.8 to 4.5

200 um b——t + C3 Proteins

Change in Length

4» Methanol Boiled Proteins

+ Water Boiled Proteins

0.0 0.5 1.0 1.5 2.0 2.5

Time (hr)

Fig.4A

U S Patent Dec. 4, 2001 Sheet 6 6f 33 US 6,326,470 B1

I .5 a:

ml 0. :3 a.

63; m . c

' O

D.

Fig.4B Time (hr)

Control Chymotrypsin Papain 4»

p >

( ugw Lu?) e185 |eugj


H/ '" (D

H u

_D m

LO. I .9

"' Q. L;

db ‘r

-- m

(km %) Mwwv

US 6,326,470 B1 U.S. Patent Dec. 4,2001 Sheet 8 0f 33

300i f _ E ____ _. s1. g 3 _._.___._ Boiled S1 l 5 .......... __ Cellulase g

g1 200._ Control 1 ~ 0.» i' I’ .J 5 |

.s -‘ i q; Protein ,' g‘ 100.. I ., t ,I - .C 1/ O .n’ "r,

0 : : r

0 0.5 1.0 1.5 2-0

Time (h)

F|q.6A

0.20

w \\ a A Control \.\51 cr: 50.15 - \ c o .9 .1 ‘

'6 l’ x LL.

5-“ B C? o.1o~- s1 Boiled _ _ _ _ _ . _ _ _ ' '_

0.05 0

Log ("?me in s)

Fig.6B


0. T T l T I N

- C _

cm

0 If) _ L_/ _l-‘

[1.

P 555 » E

P=—'__"_"'"" O Q r\ _“ F. E o3

\ '- L:

\\ L P h r:

L -- \ cu m .

g‘ 0 ' If)

(l Q_ ,

i P’) 1 .- __ O ‘ ,

<n + i O.

' j I I i O O O O O O O O n N -'

(um) q bur-n u! 8E>UDL|Q


0.6 v I T I . .

.0 (.40 I

.0 N I

HEAT-TREATED q

Change in Length (mm) D

.0 O L


+10 mM DTT

1.0

B 6. 4 2 0 0 0 0 AEEV 59.3 E mango

Time (h)

Fig.9A

25

i. 02 2mm

Time (h)

Fig.9B


- h w h n

0.8

5 A 2 0 0 0 AEEV £964 5 mmcmzo

Time (h)

Fig.1OA

20

p 5 1..

- > F O 5

1 2.5 22

2.5 2.0 1.0 1.5

Time (h)

09.105

0.5 0.0


0.5

Add Cucumber oat

0.4 ~

0.3 *

0.2 ~

4

0.1 —

Boiled walls

I I T I I I I |

Change in Length (mm)

Add Cucumber cucumber protelns

Change in Length (mm) D l\)


6_ CUCUMBER - A PROTEIN ______

i; 4_ CUCUMBER WALLS _ '5 .5 < p q

.5 5 2L. I _ Li

0 . I . 1 1 , ‘L l l . l

0102030405060 C3 Protein (pg-mL‘1)

Fig.12A

0:8 1 i I T

g CUCUMBER g 0.6- WAli * 5 CD

5 g’ 0.4 % C

2 0 0.2- -

MEAN RESPONSE (SE): OAT: 1.0 0.32 *

O0 CUCUMBER: 2.9 0.66

0.0 ‘ 0.5 ‘ 11.0 ' T5 ' 21.0 ‘ 2.5 Time (h)

Fig.12B


(L_u~%) All/013v

U.S. Patent

Activity (pm/min) Absorbance (280 nm)

Dec. 4, 2001 Sheet 18 0f 33 US 6,326,470 B1

3

2d \ 1 — \ ‘

OJ U -1_J

I I I I If I l l

O 10 2O 3O 4O Fractions

Fig.15A

3

2_-.

1_ \

0" w‘ -1 | T T ' I I I

0 1O 2O 30 4O Fractions

Fig.15B

enhancement of accessibility of cellulose by expansins

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