70

BIOTIN-BINDINGPROTEINS

[7]

with the competing sugar are pooled separately. 7 The two preparations are dialyzed exhaustively and lyophilized. Yield of nonglycosylated avidin, 24.7 mg (30%). Near-quantitative elution (54 mg, 65%) of the glycosylated forms of avidin can also be achieved. Properties of Nonglycosylated Avidin The purified nonglycosylated avidin preparation is characterized on SDS-PAGE by only one band which migrates at a position coincident with that of the low molecular weight band of Belovo avidin (Mr 15,500). The isolated protein is essentially devoid of sugar, as indicated by the lack of interaction of blotted samples with Con A or wheat germ agglutinin. The biotin-binding characteristics are virtually unchanged. Comments The nonspecific adsorption of egg-white avidin is usually attributed to two inherent characteristics of the molecule, namely, the high pI and the presence of sugar residues. For this reason, many laboratories and commercial enterprises have employed the neutral, nonglycosylated bacterial counterpart streptavidin as a substitute for the egg-white protein, despite the high cost of the bacterial protein. The availability of a nonglycosylated preparation of egg-white avidin would eliminate at least one of the abovementioned disadvantages. This is particularly important when applying avidin-biotin technology to cells that may express lectins on their surfaces. The nonglycosylated avidin has the same biotin-binding properties as the fully glycosylated form, which indicates that the sugars are not important for the biological activity. Thus, nonglycosylated avidin may eventually prove to be the preferred substitute for native avidin. 7 E f f l u e n t f r a c t i o n s are first e x a m i n e d s p e c t r o p h o t o m e t r i c a l l y a n d t h e n b y S D S - P A G E to e n s u r e t h a t o n l y t h o s e c o n t a i n i n g the n o n g l y c o s y l a t e d f o r m s o f the p r o t e i n are p o o l e d .

[7] C l o n i n g a n d E x p r e s s i o n o f A v i d i n in Escherichia coli

By GYAN CHANDRA and JOHN G. GRAY The gene coding for the biotin-binding protein avidin was isolated from a chicken oviduct cDNA, )tgtll phage library using an oligonucleotide probe derived from the published avidin protein sequence.l In t R. J. D e L a n g e a n d T. S. H u a n g , J. Biol. Chem. 246, 698 (1971).

METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

[7]

AVIDIN GENE

71

this chapter we describe the screening of the X phage library and the isolation of the gene coding for avidin protein. The successful expression of a functional form of this protein in Escherichia coli is also described. Molecular Cloning of Avidin Bacterial Strains, Phage, and Plasmids

A ~gtll chicken oviduct cDNA library was obtained from B. W. O'Malley. Bacterial strain E. coli Y10882 is used as a host to propagate the ~, phage. Escherichia coli strain JM1033 is used for propagation of the M13 mpl9 sequencing bacteriophage and for expression of the protein with plasmid pkk233-2. 4 Deoxyoligonucleotide Probe

The deoxyoligonucleotide probe is synthesized on a DNA synthesizer using phosphoramidite chemistry. 5 The probe is selected from the least degenerate codon region in the protein. The probe sequence used is determined from a tract of 8 amino acid (425-447 bp of the gene sequence) as indicated in Fig. 1. The probe is a 23-mer (23 bases) mixed-base probe and has the following sequence: 5' TTCCA(A,G)TC(A,G)TC(G,T)CC(A,G)AT(A,G)TCGTT3' Solutions and Media

20× SSC: 3 M NaC1, 0.3 M sodium citrate Denaturation: 0.5 N NaOH, 1.5 M NaCI Neutralization: 0.5 M Tris-HCI (pH 7.4), 1.5 M NaCI 5× SSCPE: 2.4 M NaCI, 0.3 M sodium citrate, 130 mM KH~_PO4, 20 mM EDTA 100× Denhardt's: 2% (w/v) bovine serum albumin, 2%, (w/v) polyvinylpyrrolidone, 2% (w/v) Ficoil (400,000 MW) Hybridization: 25% (v/v) formamide, 5× SSC, 2x Denhardt's, 150 /xg/ml sonicated salmon sperm DNA, 5 mM EDTA, 0.1% (w/v) sodium dodecyl sulfate (SDS) Wash solution: 2x SSC, 0.1% (w/v) SDS LB broth (per liter): l0 g Bacto-tryptone, 5 g yeast extract, 5 g NaCI SM buffer: 50 mM Tris-HCl (pH 7.5), 100 mM NaCI, 10 mM MgSO4, 0.01% (w/v) gelatin 2R. A. Young and R. W. Davis, Science 222, 778 (1983). 3j. Messing, Recomb. DNA Tech. Bull. (N1H Publ. No. 79-99,2) 2, 43 (1979). 4 E. Amann and J. Brosius, Gene 40, 183 (1985). 5 S. L. Beaucage and M. H. Caruthers, Tetrahedron Lett. 22, 1859(1981).

72

BIOTIN-BINDING PROTEINS i0 *

2(I *

G AAT TCC GCA AGG

60 TCC C C G Ser P r o

30 *

A G C ACA C C C G G C

70

TGT C C A

80

[7] 4(I *

50 *

CCT GCT GCA GAG ATG GTG CAC GCA ACC Met Val H i s A l a T h r 90

I00

110

CTG CTG CTG CTG CTG CTG CTC AGC CTG GCT CTG GTG GCT CCC GGC L e u L e u Leu Leu L e u Leu Leu S e t L e u A l a L e u Val A l a P r o G l y

CTC T C T Leu Ser i

120 130 140 150 * * * * G C C A G A A A G T G C T C G CTG A C T G G G A A A T G G A C C A A C G A T A l a A r g Lys C y s S e r Leu T h r G l y Lys T r p T h r A s n A s p 180 * ATC GGG GCT GTG lle G l y Ala V a l 230 * GCC ACA Ala Thr

160 * C T G G G C TCC A A C A T G L e u G l y S e r A s n Met

170 * ACC Thr

190 200 210 220 * * * * A A C AGC A G A G G T G A A T T C A C A G G C A C C T A C ATC A C A G C C G T A A C A A s n S e t A r g G l y G l u P h e T h r G l y T h r T y r Ile T h r A l a Val T h r

240 250 * * TCA AAT GAG ATC AAA GAG S e r A s n G l u Ile Lys G l u

260 270 * * TCA CCA CTG CAT GGG ACA GAA S e t P r o L e u His G l y T h r G l u

280 * A A C A C C ATC A A C A s h T h r Ile A s n

290 300 310 320 * * * * AAG A G G A C C C A G CCC ACC T T T G G C T T C A C C G T C A A T TGG Lys A r g T h r G l n P r o T h r P h e G l y P h e T h r Val A s h T r p

330 * AAG T T T T C A G A G Lys P h e Set G l u

350 360 370 * * * ACT GTC TTC ACG GGC CAG TGC TTC ATA GAC T h r V a l Phe T h r G l y G l n Cys P h e Ile A s p

390 400 * * AAG GAG GTC CTG AAG ACC Lys G l u V a l L e u Lys T h r

380 * AGG AAT GGG Arg Ash Gly

410 420 430 440 * * ~ $ A T G T G G C T G C T G C G G TCA A G T G T T A A T G A C ATT G G T G A T G A C T G G Met T r p Leu L e u A r g Set S e r Val A s n A s p Ile G l y A s p A s p T r p 460 * GTC GGC Val G l y

470 480 * * A T C A A C A T C TTC A C T CGC C T G C G C Ile A s h lle Phe T h r A r g Leu A r g

520 * AAA G C C AGC

580 CTT TGC

CTC A G A

450 * AAA G C T ACC A G G Lys A l a Thr A r g

490 500 * * ACA CAG AAG GAG TGA GGA T h r G l n Lys G l u ---

530 540 * * AAC AAT GCC GGA GTG CTG ACA CTG

550 * CTT G T G

340 * TCC A C C Ser T h r

510 * TGG CCC CGC

560 570 * * ATA T T C C T C C C A A T A AAG

590 CAA AAA

AAA AAA A

FIG. l, Complete cDNA sequence of the avidin gene. The underlined sequence indicates the signal peptide of the protein. The 8 amino acids within the box were used to select the deoxyoligonucleotide probe involved in screening the cDNA library.

[7]

AVIDIN GENE

73

Breaking buffer: 200 mM Tris-HCl (pH 7.6), 250 mM NaCI, 10 mM Mg(CH3COO)2, 5% (v/v) glycerol Nitrocellulose filters: BA/85, 0.45/zm (Schleicher & Schuell, Keene, NH) Hybridization unit: OmniBlot processing system [American Bionetics (Fisher Scientific Dist., Pittsburg, PA)]

Transfection A single colony of E. coli Y1088 cells is inoculated into 40 ml of LB broth containing 50/zg/ml ampicillin and 0.2% (w/v) maltose and incubated at 37° with vigorous shaking overnight. The culture is centrifuged at 5000 rpm for 5 min, and the bacterial pellet is resuspended in one-half the original volume (20 ml) of SM buffer. The h phage cDNA library is diluted such that 10,000 plaque-forming units (pfu)/100 mm petri plate are obtained. 2 The E. coli Y1088 cells (prepared as described above) and properly diluted phage are incubated at room temperature for 15 min to allow for phage adsorption. After adsorption, 3 ml of top agar (LB media containing 0.6% w/v agarose), prewarmed to 45 °, is added to each phage dilution and poured onto prewarmed (37 °) LB plates containing 50 ~g/ml ampicillin. Plates are allowed to solidify at room temperature for 15 min prior to incubation overnight at 37°.

Plaque Lift (Transfer of h Phage DNA onto Nitrocellulose Filters) LB plates containing phage plaques are transferred to 4 ° for 2 hr before proceeding further. Chilling the plates to 4 ° helps prevent tearing of the top agar overlay when blotting. Nitrocellulose filters (82 mm in diameter) are gently placed over the soft top agar containing the phage plaques. The position of the filter is marked using a 20-gauge syringe needle dipped in India ink. A unique asymmetric pattern is used on each filter to ensure that orientation and individual filters can be identified. Each plate containing phage is blotted in duplicate to insure against false-positive clones. The first nitrocellulose filter is left in contact with the top agar for approximately 1 min. A second or duplicate filter is allowed to remain in contact for approximately 2 min, before gently removing and placing the DNA side up on a tray containing Whatman 3MM filter paper saturated with a denaturing solution. Filters are transferred successively to neutralizing and to 2 x SSCPE solutions, keeping the filters approximately 2 min in each of these solutions. As the filters are transferred between solutions, excess liquid is removed from the underside of each filter with a paper

74

BIOTIN-BINDING PROTEINS

[7]

towel. The filters are transferred to Whatman 3MM filter paper and allowed to air dry before being vacuum dried at 80 ° for 2 hr.

Hybridization of 32p-Labeled Oligonucleotide Probe to Nitrocellulose Filters Prehybridization, hybridization, and washing of filters are done in the same Omniblot hybridization bag. Nitrocellulose filters (maximum of 20/ bag) are placed back to back in a hybridization bag, for 10 filters. For 20 filters, a sheet of mesh is placed in between as described by the manufacturer. The bag containing the filters is sealed, taking care that no mesh fibers extend into the sealing crease. A vacuum is created inside, using a 30- or 50-ml syringe, to check for leaks. After determining that no leaks are present, 60-80 ml of hybridization buffer is added to the bag containing 20 filters and incubated at 37 ° for a minimum of 2 hr. Before adding the probe, excess hybridization buffer is removed, leaving approximately two-thirds of the original volume. Approximately 1 × l06 cpm/filter of the end-labeled oligonucleotide probe 6 is diluted with hybridization buffer and added to the bag. Hybridization is accomplished by incubation at 37 ° for at least 12 hr.

Washing of Filters Washing of filters is accomplished initially using wash solution at room temperature. For 20 filters a wash volume of I liter is sufficient. The first 500-ml wash is performed at room temperature and is accomplished by passing the wash solution in a port on one side of the hybridization bag and collecting the wash in a flask as it exits the port on the opposite side. The whole process is vacuum driven. The second wash is more critical and is accomplished by washing as described above but with wash buffer prewarmed to 42 °. The entire washing procedure is monitored with a Geiger counter and is stopped when the majority of counts is effectively removed. The final critical wash may also be done in a l-liter beaker containing 500 ml of the wash buffer prewarmed to the desired temperature. Less than 5 min of exposure at this temperature is required. Filters are air dried on Whatman 3MM paper, arranged with duplicates side by side, covered with Saran wrap, and autoradiographed at - 7 0 ° with Kodak X-OMAT XAR film using an intensifying screen (Du Pont, Cronex Lightning Plus) for 24-48 hr.

Plaque Purification Signals are considered positive when signals on duplicate filters are observed to be in the same position relative to the orientation markings. 6 A. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499.

[7]

AVIDIN GENE

75

Regions approximately 1.5 mm in diameter and thought to contain positive signals are picked using a Pasteur pipet and transferred to 500/xl of SM buffer in a 1.5-ml microcentrifuge tube. After 20 min the phage suspension is used for replating each clone at a lower density of 1000 pfu/ petri plate. The replating and rescreening is repeated, thus enhancing the ratio of positive to negative clones, until 100% of the replated plaques give a positive signal. Remarks

After screening of 100,000 pfu, approximately 50 positive signals were found. The areas corresponding to 27 positive signals were picked and processed for rescreening to obtain individual pure plaques. )~phage DNA was extracted 7 and digested with EcoRI restriction enzyme to excise the insert from each clone. The EcoRI-digested DNA was electrophoresed on a 1.5% (w/v) agarose gel containing 0.7/zg/ml ethidium bromide in order to determine the size of each insert. Of the 27 clones processed, only 1 contained the entire coding sequence. The largest clone resulted in two bands of approximately 0.4 and 0.2 kbp. The two EcoRI fragments were isolated, subcloned into the M13 mpl9 phage sequencing vector, and sequenced by the Sanger dideoxy method. 8 The nucleotide sequence as well as the corresponding amino acid sequence are shown in Fig. 1. Expression of Avidin in Escherichia coli Construction o f Expression Plasmid

Utilizing the restriction enzyme information obtained from the DNA sequence analysis, it was determined that the simplest method of achieving avidin expression is to use the expression vector pkk233-2. The pkk233-2 vector contains the trc promoter (a hybrid of the trp and lac promoters with the consensus 17-bp spacing between the trp - 3 5 region and lac UV5 - l0 region) and the LacZ ribosome-binding site followed by an ATG initiation codon which is within a unique NcoI restriction site. The cloning cassette of pkk233-2 contains three unique restriction sites, NcoI, PstI, and HindIII (Fig. 2). The avidin cDNA gene has two unique restriction sites, PstI and HindIII. The PstI site of avidin is 2 bases before its ATG initiation codon and is in the same reading frame as the PstI site in pkk233-2. The HindIII site is located in the 3'-noncoding region of the cDNA gene (Fig. 3). 7 R. M. Lawn, E. F. Fritsch, R. C. Parker, G. Blake, and T. Maniatis, Cell 15, 1157 (1978). 8 F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977).

76

BIOTIN-BINDING

[7]

PROTEINS

Pvu II

602

"N~

31 bp

/

9 bp BamH I ~

\

/

I D I N "\p

EcoRI

HindIII

EcoRI .

[

Nco I ['Z-ff

Pstl

]

CAGA 'COATGGCT GOA

Met Ala

Ala

IATG GTG CAC GCA Glu

Met

Val

His

Ala

FIG. 2. Construction of the recombinant plasmid expressing the avidin protein under a trc promoter (see text). The dark area of the vector represents 529 bp of avidin in the reading frame of the promoter. Amp and Tet stand for ampicillin- and tetracycline-resistance genes, respectively. Met, Ala, Ala, and Glu, before the box, are the four amino acids added to the N terminus of the protein, whereas Met inside the box represents the first methionine (start codon) of the eDNA gene. Details of plasmid construction are described in the text.

P l a s m i d pkk233-2 is p r e p a r e d b y digesting with r e s t r i c t i o n e n z y m e s H i n d I I I a n d P s t I . A v i d i n e D N A , w h i c h had b e e n isolated from hgtl 1 b y E c o R I r e s t r i c t i o n e n z y m e d i g e s t i o n as two f r a g m e n t s (200- a n d 400-bp E c o R I f r a g m e n t s ) , is i n d i v i d u a l l y digested with P s t I (200-bp f r a g m e n t ) or

[7]

AVIDIN GENE

m

77

m

¢ = _

I

~

=

¢

I

I

100 I

200 I

m

=

300 I

400 I

500 I

600 I

bp

FIG. 3. Restriction map of avidin eDNA. The hatched box represents the coding region of the eDNA, and the open box on either side represents noncoding regions of the avidin eDNA.

HindIII (400-bp fragment). The subsequent PstI-EcoRI and EcoRIHindIII fragments are gel purified and ligated together to generate a 529-bp PstI-HindIII fragment. The PstI-HindIII fragment is ligated into pkk233-2 that had been digested with PstI and HindIII as described above. Escherichia coli JM103 cells transformed 9 with the recombinant plasmid produce individual colonies when plated on LB plates containing 50/~g/ml ampicillin. The plasmid DNA from six colonies was isolated and subjected to restriction enzyme analysis. All colonies tested contained the 529 bp PstI-HindIII avidin insert. This construction has 4 extra amino acids added to the N-terminal end of the avidin protein (Fig. 2). Procedure for Expression Escherichia coli JM103, containing the expression plasmid pkk233-2 plus the avidin gene, is inoculated into 20 ml of LB broth containing 25 ttg/ml of ampicillin and grown overnight with shaking at 37°. One-half milliliter of the culture is inoculated into 100 ml of LB broth containing 25 /~g/ml ampicillin and incubated with vigorous shaking until a Klett reading of 80 (Klett-Summerson photoelectric colorimeter) is reached. At this stage, the culture is induced by the addition of fl-isopropyl-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After a 2-hr induction at 37 °, the bacteria are pelleted by centrifuging at 5000 rpm for 5 min. The cell pellet is resuspended in 1/25 of the original volume with cold (4°) osmotic shock buffer [10 mM Tris-HC1 (pH 7.6), I mM EDTA, and 20% w/v sucrose], and the suspension is incubated for 30 min on ice. The cell suspension is again centrifuged at 5000 rpm for 5 9 j. Messing, this series, Vol. 101, p. 20.

78

BIOTIN-BINDINGPROTEINS

[7]

min. The cell pellet is resuspended in cold water (4°) at 1/25 the original volume and incubated on ice for 30 min with occasional stirring. The cell suspension is centrifuged at 8000 rpm for 10 min, and the supernatant is saved for further analysis. The cell pellet is resuspended in breaking buffer and treated with lysozyme (0.2 mg/ml) on ice for 30 rain. The lysozyme-treated cells are frozen and thawed 2 times, being careful to keep the temperature below 4°. The cell preparation is sonicated (Artek Sonic Dismembrator, Model 300) 3 times for 30 sec at 1-min intervals using a small probe at maximum setting. The cell lysate is centrifuged at 20,000 g for 30 min to clarify. DNA is removed by adding 5 mg/ml of streptomycin sulfate with stirring and subsequent incubation at 4 ° for 4-6 hr, followed by centrifugation at 20,000 rpm for 15 min. The osmotic shock and cell lysate supernatants are precipitated by adding an equal volume of 80% saturated ammonium sulfate and incubating overnight at 4°. The protein pellets obtained after centrifugation (8000 rpm for 15 min) are resuspended in 1/I00 the original volume of resuspension buffer [50 mM Tris-HC1 (pH 7.4), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Samples of each preparation (osmotic shock and cell lysate) are boiled for 3 min in Laemmli's denaturing buffer, electrophoresed on a 12.5% Laemmli gel, ~° transferred to nitrocellulose filter paper (Western blot), and probed using rabbit antiavidin antibody. 11 The Western blot can also be probed with biotinylated alkaline phosphatase ~2in order to determine whether the recombinant avidin is capable of binding biotin. To test for biotin binding by the recombinant avidin, the Western blot containing the avidin protein is first blocked by soaking in a solution of TBS [10 mM Tris-HCl (pH 7.4) and 150 mM NaC1] containing 0.2% (w/v) Tween 20 for 2 hr. Biotinylated alkaline phosphatase from Sigma (0.5 mg protein containing 5.2 tool of d-biotin per mole of alkaline phosphatase) is diluted 1:10,000 in TBST (TBS containing 0.05% v/v Tween 20) and added to the filter (about 0.5 ml of solution per cm: filter) and incubated for 1 hr at room temperature. The blot is washed 3 times for 5 min in TBST buffer. The indicator is prepared by mixing 66 /xl of nitroblue tetrazolium (NBT) substrate with 10 ml of alkaline phosphatase buffer [100 mM Tris-HC1 (pH 9.5), 100 mM NaC1, 5 mM MgC12] and 33/xl of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate as described in the manufacturer's instructions (Promega Biotech, Madison, WI). The indicator is added and the color allowed to develop for l0 min before l0 U. K. Laemmli, Nature (London) 227, 680 (1970). i1 Z. Wojtkowiak, R. C. Briggs, and L. S. Hnilica, Anal. Biochem. 129, 486 (1983). tz y . Hiller, "J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987).

[7]

AVIDIN GENE

79

being stopped by washing with several changes of water at 5-min intervals. The results of the Western blots (antibody probe and biotin probe) demonstrate that avidin is expressed in E. coli, is secreted into the periplasmic space, and is still capable of binding biotin. Concluding Comments Using an oligonucleotide probe to the published protein sequence of avidin, we were able to obtain a cDNA clone from a chicken oviduct eDNA library encoding for the entire protein. Gope et al. 13 have also reported the molecular cloning of chicken avidin cDNA, and their published sequence is virtually the same as that reported here for the 596-bp clone. The clone of 596 bp contained the published protein sequence of amino acids plus an additional 24 amino acids on the N-terminal end thought to be a signal peptide, which is probably responsible for the avidin protein being found in the periplasmic fraction (osmotic shock) of E. coll. The avidin-biotin studies, performed for the expressed protein secreted in the periplasm of E. coli, revealed that recombinant avidin is still capable of biotin binding. Therefore, it will now be possible to study the mechanism responsible for its high-affinity binding to biotin. In addition, cloning of the gene gives the opportunity (1) to tag genes of interest with avidin for study in vivo and (2) to make hybrid proteins through genetic engineering that can be easily purified using the avidin-biotin interaction. Acknowledgments We are thankful to Ms. Jennifer Lorenz and Mr. Stephen Haneline for their valuable assistance on this project.

13 M. L. Gope, R. A. Kein~men, P. A. Kristo, O. M. Conneely, W. G. Beattie, T. ZaruckiSchulz, B. W. O'Malley, and M. S. Kulomaa, Nucleic Acids Res. 15, 3595 (1987).