[29]

RECOMBINANT NSF FROM E. coli

309

point using a crude whole-tissue extract as starting material (where no NSF activity can be detected). After the sedimentation on glycerol gradient, we found that most of the inhibitory activity is removed and that the NSF peak could be followed during the final purification steps. Therefore, it is likely that mammalian NSF in tissue extracts can be purified using our procedure with minor modifications.

[29] E x p r e s s i o n a n d P u r i f i c a t i o n o f R e c o m b i n a n t N-Ethylmaleimide-Sensitive Fusion Protein from Escherichia coli B y DUNCAN W . WILSON a n d JAMES E. ROTHMAN

The N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) is a homotetramer of 76 kDa, initially purified from Chinese hamster ovary (CHO) cells t and essential for the fusion of transport vesicles with their cognate acceptor membrane 2 at many stages in the secretory and endocytic pathways. Studies in vitro and in vivo have implicated mammalian NSF and Sec 18p, the Saccharomyces cerevisiae homolog,3 in endoplasmic reticulum (ER)-to-Golgi traffic, 4 in at least two stages of intra-Golgi transport, and in endocytosis.5 For an overview, see Wilson et al. 6 and references therein. Here we describe techniques for the expression and purification of large quantities of active, recombinant CHO cell NSF from the bacterium Escherichia coll. Availability of unlimited amounts of this protein, and the opportunity to prepare genetically modified derivatives, will greatly facilitate analysis of the biochemical and molecular properties of NSF.

M. R. Block, B. S. Glick, C. A. Wilcox, F. T. Wieland, and J. E. Rothman, Proc. Natl. Acad. Sci. USA 85, 7852 (1988). 2 V. Malhotra, L. Orci, B. S. Glick, M. R. Block, andJ. E. Rothman, Cell54, 221 (I988). 3 D. W. Wilson, C. A. Wilcox, G. C. Flynn, E. Chen, W.-J. Kuang~ W. J. Henzel, M. R. Block, A. Ullrich, and J. E. Rothman, Nature (London) 339, 355 (1989). 4 C. J. Beckers, M. R. Block, B. S. Glick, J. E. Rothman, and W. E. Balch, Nature (London) 339, 397 (1989). s R. Diaz, L. S. Mayorga, P. J. Weidman, J. E. Rothman, and P. D. Stahl, Nature (London) 339, 398 (1989). 6 D. W. Wilson, S. W. Whiteheart, L. Orci, and J. E. Rothman, Trends Biochem. Sci. 16, 334 (1991).

METHODS IN ENZYMOLOGY, VOL. 219

Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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IDENTIHCATION OF TRANSPORT INTERMEDIATES

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Construction of Recombinant Expression System for NSF We chose to express a derivative of NSF bearing a carboxy-terminal epitope "tag" because of the lack of anti-NSF antibodies suitable for immunoprecipitation or Western blotting. Cleavage of the DNA encoding NSF with the restriction endonuclease NheI excised the last six codons of the NSF gene (see Fig. 1). A synthetic oligonucleotide was used to restore these 6 codons and then extend the gene by 10 additional codons corresponding to the amino acids EQKLISEEDL (the sequence of the new 3' end of the NSF open reading frame was confirmed by DNA sequence analysis). The sequence EQKLISEEDL is recognized by a mouse monoclonal IgGt antibody termed 9E 10, raised against peptides derived from the predicted amino acid sequence of human c-myc, 7 and which has previously proved successful as an epitope tag. s We refer to the recombinant NSF encoded by this gene as NSF/Myc. To test that the NSF/Myc protein is active we prepared cytosol from a S. cerevisiae secl8-1 strain bearing the NSF or NSF/myc genes expressed from the GALI-IO promoter. Normally, cytosol from secl8-1 mutant S. cerevisiae has no detectable NSF activity when tested in an NSF-dependent transport assay3; however, extracts from mutants expressing the NSF or NSF/myc genes contained levels of NSF activity comparable to wild-type SEC18 yeast (data not shown). The "tagged" NSF/rnyc gene therefore has the capacity to encode an active protein in this eukaryote. We then attempted to achieve expression of active protein in E. coli, in order to maximize the amount of NSF/Myc that could be recovered. An XbaI-XhoI DNA fragment, containing the entire NSF/myc gene, was ligated with the bacterial expression plasmid pTTQ 18,9 previously cleaved with the restriction enzymes XbaI and Sail. The resulting plasmid, pTTQNSF/myc, is shown in Fig. 1. The XbaI restriction site is the point of translational fusion between the pTTQ18 vector and a linker contiguous with the CHO cell-derived DNA fragment bearing the NSF gene. As shown in Fig. 1, this fusion results in an amino-terminal extension of 22 amino acid residues (Met-Asn-Ser . . . Ser-Ala-Lys) prior to the methionine residue proposed by Wilson et al. 3 to be the true amino terminus of NSF on the basis of position and ATG context. The E. coli strain NM522 (9) was transformed with pTTQNSF/myc and transformants selected on plates of Luria broth (LB; 10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaC1, pH 7.2) supplemented with 50 7 G. I. Evan, G. K. Lewis, G. Ramsay, and M. J. Bishop, Mol. Cell. Biol. 5, 3610 (1985). 8 S. Munro and H. R. B. Pelham, Cell48, 899 (1987). 9 M. J. R. Stark, GeneSl, 255 (1987).

RECOMBINANT NSF FROM E. coli

[29]

311

Encodes the c-myc D e r i v e d Amino Acid Sequence C o r r e s p o n d i n g to an Epitope Restores the C-Termlnal

for the Monoclonal

Sal I/Xho I J u n c t i o n

IgG 9El0

of Ollgonucleotlde and pTTQI8 Polyllnker

Region of the NSP Protein

l

tl

... Ser Pro Leu Asp Phe Asp Giu Gln Lys Leu lle Set GIu Glu Asp Leu *** • ,CTAGC CCC CTG GAC TTT GAT GAG CAG AAA CTG ATC AGC GAA GAG GAC CTG TGA CTCGAC.. , • ,GATCG GGG GAC CTG A A A CTA CTC GTC TTT GAC TAG TCG CTT CTC CTG GAC ACT GAGCTG,,,

i i

U

Nhe I Junction of NSF Gene and

STOP

O l l g o ~

P 01

/ Polylinker of pTTQI8 Vector

I

Met Asn Ser Set Ser Val Pro Gly Asp Pro Leu A s p Val Asp Ser Glu Pro Asp Val Ser Ala Lys Met..• GCG ATG AAT TCG AGC TCG GTA CCC GGG OAT CCT CTA GAC GTC GAC TCG GAA CCG GAC GTG TCC GCG AAG ATG... CGC TAC TTA AGC TCG AGC CAT GGG CCC CTA GGA OAT CTG CAG CTO AGC CTT GGC CTG CAC AGG CGC TTC TAC,..

I

I

Xba I Junction

l Cloned Fragment C o n t a i n i n g NSF Gene

FIO. 1. Structure of the IPTG-inducible transcribed region of plasmid pTTQNSF/myc, used for expression of NSF/Myc in E. coli. Open bars, tac promoter (Pro) and ribosomal RNA transcriptional terminator (rrnB); solid bars, polylinker regions derived from parent vector pTTQ18; hatched bar, DNA fragment encoding the NSF gene up to the NheI restriction site; horizontally striped bar, region derived from synthetic oligonucleotides. Direction of transcription is shown by a horizontal arrow above the NSF/myc gene. Note particularly the XbaI restriction site (point of fusion between DNA containing the NSF/myc gene and that from pTTQ18), the NheI restriction site (which marks the point at which the C terminus of NSF becomes encoded by a synthetic oligonucleotide), and the XhoI]SalI junction (where the DNA encoding NSF/Myc rejoins that of vector pTTQ 18).

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#g/ml ampicillin (sodium salt), 5 g/liter dextrose (LBAD medium) and 2% (w/v) agar. pTTQNSF/myc transformants were grown using LBAD broth (except when inducing plasmid expression). Transformants rapidly became inviable when stored on LBAD-agar plates, even at 4 °. Consequently, we store pTTQNSF/myc transformants frozen at - 70 ° in LBAD containing 12.5% (w/v) glycerol. Preparation of Bacterial Extracts with NSF Activity To prepare bacterial extracts containing active NSF/Myc, 50 ml of LBAD broth is inoculated with cells scraped from the surface of a frozen aliquot of pTTQNSF/myc transformants. The culture is shaken overnight at 37 °, then 10 ml is used to inoculate 1 liter of LBAD broth, prewarmed to 37 ° . This culture is shaken vigorously at 37 ° to ensure efficient aeration (typically, we shake 500 ml of broth in a 2-liter conical flask) until mid-log phase, when the optical density achieves 0.6, measured at a wavelength of 660 nm (under these conditions, this takes between 2.5 and 3 hr). Cells are pelleted at 6000 rpm for 10 rain in a Sorvall (Norwalk, CT) GS3 rotor at room temperature and the pellets completely resuspended in 500 ml of prewarmed LB. The purpose of this wash is to remove dextrose from the cells, to ensure efficient induction. After pelleting again, the cells are resuspended in 500 ml of prewarmed LB supplemented with 50/lg/ml ampicillin and 0.5 m M isopropyl-fl-D-thiogalactopyranoside (IPTG). We dispense 250 ml of culture to each of two 2-liter conical flasks and shake vigorously at 37 ° for 3 hr. From this point, all procedures are at 4 °, using prechilled bottles, solutions, and centrifuge rotors. Cells are pelleted as before, then washed by resuspension in 50 ml breaking buffer containing 100 m M N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH, pH 7.8, 5 mMATP, I mMethylene glycol-bis(fl-aminoethylether)-N,N,N',N'tetraacetic acid (EGTA), 500 m M KCI, 5 m M MgC12, 1 rrLMdithiothreitol (DTT), 1 m M phenylmethylsulfonyl fluoride (PMSF). Cells are pelleted again, resuspended using 25 ml breaking buffer (yielding a final volume of approximately 35 ml), and disrupted by two passages through a prechilled 40K French pressure cell (SLM AMINCO Instruments, Urbana, IL), at 16,000 psi. The lysate is centrifuged for 30 min at 15,000 rpm in a Sorvall SA600 rotor and the low-speed supernatant (LSS) carefully decanted from the pellet. We typically recover 26-28 ml of LSS, at a protein concentration between 8 and 10 mg/ml. At this point, NSF activity can be measured directly in the LSS (see the next section), and the polypeptide readily detected by Coomassie Brilliant blue staining when 10 pg of LSS is resolved by sodium dodecyl sulfate-

[29]

RECOMBINANTNSF FROME. coli

313

polyacrylamide gel electrophoresis (SDS-PAGE) using a 7.5% (w/v) polyacrylamide gel; NSF/Myc migrates as an 85K band comprising approximately 0.5% of total protein (estimated by eye). We have found it is most useful to compare the LSS extract of a pTTQNSF]myc transformant with that prepared from cells containing the pTTQ18 vector. In the latter extract, the 85K band is absent, as is NSF activity (see below). If 1 pg of LSS is electrophoresed as above, then Western blotted, the 85K band can be shown to cross-react with the 9El0 anfi-myc monoclonal antibody (data not shown). No other E. coli protein has been observed to cross-react with the 9El0 antibody under these conditions. N S F Activity D e t e c t e d in C r u d e Extracts N-Ethylmaleimide-sensitive fusion protein activity can be detected within the crude LSS extracts by assaying between 20 and 100 ng of LSS protein in a 25-#I NSF-dependent Golgi transport assay (see Block et al.' for assay conditions); however, transport activity is greatly inhibited if more than 1 pg of LSS is added to the assay. The LSS can be stored at - 7 0 ° following rapid freezing in liquid nitrogen, and on thawing shows little loss of activity. That the activity measured in this extract is indeed due to expression of active CHO cell NSF, and not to a nonspeeific effect of the lysate, was confirmed in a number of ways. First, activity always copurified with the NSF/Myc polypeptide (see below). Second, activity is absent if LSS is prepared from E. coli that carry the pTTQ 18 vector but not the NSF/myc gene (Fig. 2) or in LSS from pTTQNSF/myc transformants that have been "mock-induced" using 0.5% (w/v) dextrose rather than 0.5 m M IPTG (data not shown). Third, treatment of the LSS with 1 m M NEM or incubation at 37 ° in the absence of ATP, conditions known to inactivate CHO cell NSF, abolish NSF activity in these extracts (Fig. 3). Last, LSSderived NSF activity is inhibited by the anti-CHO NSF monoclonal IgM antibody 4A6, which inhibits CHO cell NSF activity in vitro.~ A control IgM monoclonal antibody fails to affect LSS NSF activity (see Fig. 4). Sensitivity to inhibition by 4A6 appears to be a highly specific characteristic of CHO NSF activity; 4A6 has no effect on the activity of Sec 18p (data not shown), even though it shares 49% primary sequence identity with NSF (Wilson et al.3). Purification of N S F / M y c from Escherichia coli We have found that the chromatographic properties of bacterially expressed CHO cell NSF/Myc are very different from those of the protein obtained directly from CHO cells (for reasons discussed below). We have

314

IDENTIFICATION OF TRANSPORT INTERMEDIATES

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E 1: 0 a. m c:

700 5o0.

3oo~ II) "0

100 -

if) Z 0

10

20

30

40

SO

60

70

80

90

Bacterial extract (no) FIG. 2. NSF activity is found only in bacterial extracts if the NSF/myc gene is present in the expression vector. Low-speed supernatant extracts were prepared from IPTG-induced E. coli NM522 transformants (see text) and assayed in 25-/tl NSF-dependent transport assays. Assays were in duplicate and the mean value plotted. A background (NSF/Mye-independent) signal of 343 counts per minute (epm) has been deducted from the data. (e) E. coli transformed with pTTQNSF/myc;(I1) E. coli transformed with pTTQ18 vector.

therefore developed a purification scheme that differs markedly from that described by Block et aL I The characteristics of the purification are listed in Table I, and the appearance of the active fractions following SDS-PAGE on a 7.5% (w/v) polyacrylamide gel is shown in Fig. 5. First, an LSS is prepared exactly as described above, then clarified by centrifugation at 50,000 rpm for 1 hr in a Beckman (Palo Alto, CA) Ti 60 rotor. The high-speed supernatant (HSS) is recovered by decanting and pipetting, carefully avoiding the loose gelatinous membrane fraction that overlays a firmer, high-speed pellet. The protein concentration of the HSS is corrected to 6 mg/ml using breaking buffer, then a solution of polyethylene glycol 4000 [made to 50% (w/v) in water] slowly added to a final concentration of 10%, while stirring continuously at 4°. After stirring for a further 30 min the mixture is centrifuged at 5000 rpm for 20 min in a SorvaU SA600 rotor, the supernatant discarded, and the pellet gently resuspended in a Dounce homogenizer (Wheaton, Millville, NJ) in 10 ml of Q200 buffer [20mM piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES)-KOH, pH 7.0, 2 mM MgCl2, 2 mM DTT, 0.5 mM ATP, 10% (w/v) glycerol, 200 mM KC1].

RECOMBINANTNSF FROM E. coli

[29]

315

600

E

5oo

~.

400

o Q. u)

300

o. ¢)

.., c "0 t-

"f / . ,/. / /' / f/ / / A. r l l l l l l ¢ / / / / / J

200

, 1 1 / / i , , 1 1 1 1 1 ,

!

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/

t

!

,

~ 1 1 / / I / I ] 1 1 1 ] . " ] l l ] l l f 1 1 1 1 1 / I I I I I 1 ~

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-

r l l l l l l , . 1 1 1 1 ,

0

(i) Z

NEM Treated

Mock Treated

Warmed Warmed (No ATP) (+ATP)

FIG. 3. Biochemical properties of bacterially expressed NSF/Myc. N-Ethylmaleimide sensitivity studies: LSS extracts were diluted 500× into 50 mM HEPES-KOH, pH 7.5, 50 mM KCI, 5 mM ATP, 5 mM MgC12, 1% (w/v) polyethylene glycol 4000, 10 mg/ml soybean trypsin inhibitor, to deplete dithiothreitol (DTT). Diluted LSS was incubated with 1 mM NEM (from a freshly prepared 50 mM stock solution) for 15 min at 4", then excess NEM quenched by incubation with 2 mM DTT for 2 rain at 4". In a control incubation, diluted LSS was mock treated using 1 mM NEM that had been preincubated with 2 mM DTT. ATP stability studies: LSS extracts were diluted 500X into 50 mM HEPES-KOH, pH 7.5, 50 mM KC1, 2 mM DTT, 5 mM MgCl~, I% (w/v) polyethylene glycol 4000, l0 mg/ml soybean trypsin inhibitor to deplete ATP. The depleted sample was incubated at 37" for 30 min, then returned to ice and ATP added to a final concentration of 1 raM. In a control incubation, the sample was mixed with ATP (to 1 raM) prior to 37" treatment. Fifty nanograms of the treated samples and controls were tested for NSF activity in duplicate, and the mean plotted. A background signal (see Fig. 2) of 685 clam has been deducted from the data.

W h e n fully resuspended, the sample is loaded at 0.5 m l / m i n onto a 20 × 3 c m c o l u m n containing 60 m l Q Sepharose fast-flow (Pharmacia, Piscataway, N J) previously equilibrated with Q200 buffer (in pilot trials we found that, under these conditions, A T P fails to bind to this resin at KCI concentrations greater t h a n 160 m M ) . T h e c o l u m n is washed with 2 vol of Q200 buffer at 0.5 m l / m i n , then b o u n d N S F / M y c eluted by washing with 2 vol o f Q400 (identical to Q200 buffer, but containing 400 m M K C 1 ) at 1.0 m l / m i n . As well as yielding a substantial e n r i c h m e n t for N S F / M y c relative to other proteins (Table I), this rapid a n d simple c o l u m n step is essential for the preparation of a sample sufficiently free o f lipid a n d nucleic acids to permit anion-exchange c h r o m a t o g r a p h y using fast protein liquid c h r o m a tography (FPLC). The Q400-eluted material is mixed with an equal vol-

316

IDENTIFICATION OF TRANSPORT INTERMEDIATES A

[29]

400

E O

300

1::

o

==

200 100

4)

"o p.

o.

0

"o u)

w

;

0.0

- - t

i. . . . .

0.2

0.4



.....!

.... •

0.6

i

0.8

....

,

F

1.0

IgM preincubated with bacterial extract (pg) FIG. 4. NSF/Myc Activity in bacterial extract is inhibitedby a monoclonal antibody raised against CHO cell NSF. Low-speed supernatam (56 rig) was added to an NSF-degendent transport assay cocktail such that the final KCI concentration was 65 mM (necessary to maintain the solubility of the 4A6 IgM; see Ref. 1). Increasing amounts of the 4A6 antibody (0) or a control IgM ~ were added and the mixture incubated for 15 rain at 4". Assays were in duplicate and the mean value plotted. A background (NSF/Myc-independent) signal of 656 corn has been deducted.

TABLE I PURIFICATIONOF NSF/Myea

Sample 100,000 g supernatant 1096 polyethyleneglycol (PEG) precipitation Fast Flow Q, 400 mMKC1 batch elution Mono Q pool

Total protein (rag)

Total activity (units)

Specific activity (units/mg)

Enrichment

Yield (%)

213 50.9

2.8 × I0 '° 1.9 × 101°

1.3 X 10s 3.7 × l0 s

1 2.8

100 67.9

9.5

1.7 X 10l°

1.8 )< 109

13.8

60.7

0.9

0.76 x 10t°

8.4 X 109

64.6

27.1

a Enrichment and yield are soecified relative to the high-s~ed supernatant (HSS). Activity units for each sample are counts per minute of N-aeetyl[3H]glucosamine that became incorporated into vesicular stomatitis virus G protein as a result of addition of that sample to an NSF-dependent in vitro transport reaction. Counts IX* minute due to NSF-independent incorporation (387 cpm in these assays) were deducted from the data. All activity estimates were made after dilution of sample into the linear range for assay.

[29]

RECOMBINANTNSF FROME. coli 1

234

317

5 "91--200 116

~97 "~I-"66 45

~---31 "~"22 Fro. 5. Sodium dodecylsulfategel electrophoresisof samples recoveredat each stage of the NSF/Myc purification.The followingamounts of total protein from each stage of the purification were subjected to SDS gel electrophoresis on a 7.5% (w/v) polyacrylamidegel, then visualized by staining with CoomassieBrilliant blue. Lane 1, 20 #g High-speedsupernatant (HSS); lane 2, 7/tg 10% (w/v) PEG precipitate; lane 3, 5/~g Fast Flow Q 400 mM KC1 elution;lane 4, 0.3/tg Mono Q FPLC pool; lane 5, 0.3 ]tg NSF purifiedfrom CHO ceilsby the method of Block et aL t Note the increasedsize of the NSF/Myc protein, which is 32 amino acids larger than NSF purified from CHO cells. Arrows indicate the migration of standard proteins of known molecularweight(× 10-3). ume of Q0 buffer (containing no KC1) to dilute the KC1 to a concentration of 200 mM, then concentrated using an Amicon (Danvers, MA) concentrator and YM30 membrane. After concentration to 30 ml or less, the sample is loaded onto a 1-ml Mono Q FPLC column (preequilibrated in Q200 buffer) at 0.5 m l / m i n and the column washed with 5 vol of Q200 at 0.5 ml/min. Bound protein is eluted with a 20-ml linear gradient from 200 to 500 m M KC1, while maintaining other buffer conditions constant, and 0.5-ml fractions collected. The NSF activity elntes at 300 m M KC1, and 7.5% (w/v) SDSPAGE shows the activity coincident with an 85K band that, following blotting, cross-reacts with the 9El0 antibody (data not shown). Conservative pooling of fractions at this stage is important to avoid contamination of NSF/Myc with a 50K protein that elutes from the column at a slightly higher KC1 concentration. We normally recover two pools (which contain similar total amounts of NSF/Myc): the first is from the peak activity fractions and appears homogeneous when electrophoresed and visualized by Coomassie Brilliant blue staining; the second is derived from side

318

IDENTIFICATION OF TRANSPORTINTERMEDIATES

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fractions, and contains the 50K contaminant (present at a concentration similar to NSF/Myc). Although we use the pure fraction for most purposes we have encountered no problems using the less pure pool. Pooled fractions are dialyzed against 1000 vol of NSF buffer [50 mM HEPES- KOH, pH 7.5, 50 mMKC1, 0.5 mMATP, 5 mMMgC12, 2 mMDTT, 10% (w/v) glycerol] at 4 ° for 2 hr using Spectrapor 2 dialysis tubing, then aliquots frozen in liquid nitrogen for storage at - 7 0 °. A standard 25-/~1 NSFdependent in vitro transport assay is saturated by 20 ng of NSF/Myc purified in this manner (data not shown). Of great advantage in the purification of NSF/Myc was the finding that this protein remains bound by Q Sepharose (and also diethylaminoethyl (DEAE)-ceUulose; data not shown) at high salt concentrations (up to 300 m M KCI) at a pH of 7.0. In contrast, at pH 7.0 NSF purified from CHO cells does not bind DE-52 resin even at 100 mM KCI. t In subsequent studies, Tagaya et al.~° have used the pTTQ 18 expression vector to prepare NSF that does not contain the C-terminal epitope tag EQKLISEEDL. This protein still binds to Q Sepharose but can be eluted at much lower KC1 concentrations than NSF/Myc. We believe it likely that the aminoterminal extension present in both forms of bacteriaUy expressed NSF (and which contains five negatively charged amino acids; see Fig. 1) results in the recombinant protein having an increased affinity for anion-exchange resins. Much higher affinity then results from addition of the myc epitope tag, which contains four acidic residues, three of them in tandem adjacent to the most C-terminal amino acid of the protein. Use of this epitope may therefore be advantageous for purification of the recombinant protein, dependent on the context in which it is placed. Acknowledgments We thank T. Silhavy and members of the Silhavy laboratory for the use of their French press, and M. Wiedmann for helpful discussions. ~0M. Tagaya, D. W. Wilson, M. Brunner, N. Arango, and J. E. Rothman, submitted for publication.

Expression and purification of recombinant N-ethylmaleimide-sensitive fusion protein from Escherichia coli.

[29] RECOMBINANT NSF FROM E. coli 309 point using a crude whole-tissue extract as starting material (where no NSF activity can be detected). After...
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