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Purification and characterization of heat-modifiable protein from the outer membrane of Escherichia coli
FEB$ LETTERS.
Volume 41, n ~mbei 2
"
PURIFICAT PROTEIN R. A. F, REITHMEIER and P . D . BRAGG D e p a r t m e n t ¢,f B "~¢~
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, :U t ~ s i
t y o f ~ t i ~ h ~Columbta l
VanCou~er, B.C. :Ca,~ada Received 8 March t 9 7 4
1. Introduction The outer membrane of Gram.negative bacteria is unique in that it contains lipopolysaccharide (LPS) and is tightly associated with peptidoglycan [1]. Phospholipid is not required for the structural integrity of this membrane [2], indicating that the fluid mosaic model of Singer ~nd Nicolsoa [3] may not adequately describe it~ stracture. While the inner, cytoplasmic membrane of E. coli is composed of a large number of protein components, the outer membrane contains less than a dozen major proteins [1 ]. The unique properties and ~he simpl.* protein composition of, the outer n :~mbrane make it an interesting system for study. The properties of only a few outer membrane proteins from E. coli have been described. The amino acid sequence of the lipoprotein which, attaches the outer membrane to the peptidoglycan ha:; been determined [4]. The protein receptors for colicin E3 [5], and for both phage T5 and colicia M [6] have recently been purified. Phospholipave A t activity has been localized to the outer membrane [7]. In this communication, we report the purification and preliminary characterization of a major protein component of the outer membrane o r E . coll. This protein may be involved in maintaining the shape of the bacterium [R]. Protein B [t}] with an apparent molecular weight of 28 500 is quantRatively converted to a form B* with a molecular weight of 33 400 upon heating in sodium dod,;cyl sulfate (SDS)-containing solutions at temperatures higher than 50°C.Cyanogen bromide cleavage an~ N.terminal
analysis of B a n d B* showe~l tha" they were the same protein. The conversion aliowed the isolaiioil of protein B* h~ a homogeneous form.
2. Exlm,-imental 2.1. Purification o f membrane prote:'ns The ceU wall layer, com~sting o f outer membrane and peptidoglycan was prepared from E. o, t, N.~C 482 by solubilization of the inner membrane ~,.]th Triton X-I 14 as previously• d e s c r i e d [ 10]. ?:o tein.~ were sequer, tially extracted 1', ~m this frac~ior b~: treatment with 0.5% SDS at 7°C for l hr, f¢,llcwvd by extraction with 2.5% SDS at lO0°C for !~ mi~,~. The membranes were centrifuged:at400 ~ g t O r . : . 30 rain after each extract~onistep, to.produce~twC . " . extracts, 1 and 2, and a ~esidueof pC.ptidoslycan. The extracts were dialyz~,d a ~ s t distit~, water a t : room temperature tbr 24 lxr ~md then=a~ 4°C.for ' S days, to remove SIX.q, 10g (wet weigh0 vffrozen cells produced an average yield of 2 0 a n d 35 mg o f protein in exta~ts 1 anti 2, respectively. In atypicai experiment, 40 m8 of extrttct I was dissolved in l 0 ml of 0.1 M s o d l m phosphate ~uffer, pH 7.2, containing 1% SDS and OA%/~-n~rcap~oethanol(ME), and incuba*.ed at 37°C for 1 hr...The clear sol ~ i o n was-applied to a column of Sephadex GIO0 (2.~ X 40 cm) connected in series to a column of Sepha:~ .~e 6B of the s~tr-,~ dimensions. The Proteins ¢~re eluted at roc ~.~t ~ m p c r a ~ e w ! t h " sodium phosphate buffe~ pH"L2~¢ontaint~ 1% ~
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Volume 41, n ~mbei 2
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PURIFICAT PROTEIN R. A. F, REITHMEIER and P . D . BRAGG D e p a r t m e n t ¢,f B "~¢~
~
, :U t ~ s i
t y o f ~ t i ~ h ~Columbta l
VanCou~er, B.C. :Ca,~ada Received 8 March t 9 7 4
1. Introduction The outer membrane of Gram.negative bacteria is unique in that it contains lipopolysaccharide (LPS) and is tightly associated with peptidoglycan [1]. Phospholipid is not required for the structural integrity of this membrane [2], indicating that the fluid mosaic model of Singer ~nd Nicolsoa [3] may not adequately describe it~ stracture. While the inner, cytoplasmic membrane of E. coli is composed of a large number of protein components, the outer membrane contains less than a dozen major proteins [1 ]. The unique properties and ~he simpl.* protein composition of, the outer n :~mbrane make it an interesting system for study. The properties of only a few outer membrane proteins from E. coli have been described. The amino acid sequence of the lipoprotein which, attaches the outer membrane to the peptidoglycan ha:; been determined [4]. The protein receptors for colicin E3 [5], and for both phage T5 and colicia M [6] have recently been purified. Phospholipave A t activity has been localized to the outer membrane [7]. In this communication, we report the purification and preliminary characterization of a major protein component of the outer membrane o r E . coll. This protein may be involved in maintaining the shape of the bacterium [R]. Protein B [t}] with an apparent molecular weight of 28 500 is quantRatively converted to a form B* with a molecular weight of 33 400 upon heating in sodium dod,;cyl sulfate (SDS)-containing solutions at temperatures higher than 50°C.Cyanogen bromide cleavage an~ N.terminal
analysis of B a n d B* showe~l tha" they were the same protein. The conversion aliowed the isolaiioil of protein B* h~ a homogeneous form.
2. Exlm,-imental 2.1. Purification o f membrane prote:'ns The ceU wall layer, com~sting o f outer membrane and peptidoglycan was prepared from E. o, t, N.~C 482 by solubilization of the inner membrane ~,.]th Triton X-I 14 as previously• d e s c r i e d [ 10]. ?:o tein.~ were sequer, tially extracted 1', ~m this frac~ior b~: treatment with 0.5% SDS at 7°C for l hr, f¢,llcwvd by extraction with 2.5% SDS at lO0°C for !~ mi~,~. The membranes were centrifuged:at400 ~ g t O r . : . 30 rain after each extract~onistep, to.produce~twC . " . extracts, 1 and 2, and a ~esidueof pC.ptidoslycan. The extracts were dialyz~,d a ~ s t distit~, water a t : room temperature tbr 24 lxr ~md then=a~ 4°C.for ' S days, to remove SIX.q, 10g (wet weigh0 vffrozen cells produced an average yield of 2 0 a n d 35 mg o f protein in exta~ts 1 anti 2, respectively. In atypicai experiment, 40 m8 of extrttct I was dissolved in l 0 ml of 0.1 M s o d l m phosphate ~uffer, pH 7.2, containing 1% SDS and OA%/~-n~rcap~oethanol(ME), and incuba*.ed at 37°C for 1 hr...The clear sol ~ i o n was-applied to a column of Sephadex GIO0 (2.~ X 40 cm) connected in series to a column of Sepha:~ .~e 6B of the s~tr-,~ dimensions. The Proteins ¢~re eluted at roc ~.~t ~ m p c r a ~ e w ! t h " sodium phosphate buffe~ pH"L2~¢ontaint~ 1% ~
:~fibso}.bi~fiCe :measuredla~t 2 8 0 n m . : . F , . a c i i 0 n s w e , e : : ~ .--: : - .-.:. (:]::: ; - -- :.: i ~ s a y e d 5 o r : p r 0 t e i n : a n d : l i p o p o l y g a echai-~de ( a s . 2 * e t,b. ." ' . ?:3-de0X),0ct0hicabid (KDO))?acco*d~ngi'm:-t~.;[i il] " . ; } ~ d 0 s b i 0 . r n : . . [ l 2 ] , ,especti'~(e]y,:C:oMmn s a m p l e s : -- " •-. ( 5 0 -arid-1:.00 p l ) - w e ~ - : k n c u b a t e d w i i h a f e w .c~ysta..]g .. : . :"-:eJ'-~rea a n d ~E::(t~ma i c m a c ; z n . t r a f i o n , O .i %)-for:: ~ : - . ..:.1~. nun.a=t 0 7 C a n d -run on: ] 0 % S D S - p o t y a c ~ y l a m ~ . d . e gelS a s d e s . e r i b e d = b e ~ o l e - I 9 1 t . ~ • ..- " " =i - i ._ • . i. -.:. T h e f r a c J i o n s c e n t ~ i n i n g p r o t e i n B w e ~ e p 0 o l . e d : , , d i a l y z e d ag~nst_dist:ilte d vzal er, a n d ! y o p h i l i z e d. O n e - 1 r e ! f o r t h i s : f r a c t i o n Was sa~:,ed f o r ¢ h a r a c t e S z a t i o n . " " ::The o t h e } h a l f w a s d i ~ s o ] v e d in o . a M s o d i u m p h o s p h a t e b ~ r f e r ; # ~ 7 2 , c o m b i n i n g a'~ S D S a n a 0.~ .
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~ ~ 2 • a . ~ . . • .. • . ~i.~=~-~m . .- . r~g. a: Efa'ee.~0f lmafi~-, o n m ~ r a d o n o f ~ r m e i ~ B ~ SDS polymaxyla.m]de_gels. A : Densitometez.sea~aing :g~aees o f
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