[6] Nonglycosylated avidin

68

BIOTIN-BINDING PROTEINS

[6]

[6] N o n g l y c o s y l a t e d A v i d i n

By YAFFA HILLER, EDWARDA. BAYER, and MEIR WILCHEK We have recently found ~ that a commercial preparation of egg-white avidin2 contains about 30% of a nonglycosylated form of the tetrameric protein. During the course of studies on this protein, we developed a method for the isolation of the nonglycosylated tetramer based on the removal of glycosylated forms 3 using appropriate lectin columns. Since avidin is known to contain both mannose and N-acetylglucosamine residues, 4 affinity columns containing concanavalin A (Con A) or wheat germ agglutinin can be used for this purpose. Con A is the lectin of choice because it is relatively inexpensive and because immobilized forms are obtainable from a variety of commercial sources. Even though no method is currently available for the experimental deglycosylation of avidin, 5 we describe here a method for the separation and isolation of the fully nonglycosylated tetramer from mixtures of glycosylated and partially nonglycosylated forms of the protein. This method will undoubtedly be useful when deglycosylation procedures are eventually described. 6

J Y. Hiller, J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987). z Batch 1283, Belovo Soc. Coop. (Bastogne, Belgium). 3 Glycosylated forms of avidin refer to tetramers containing either fully glycosylated monomers, monomers bearing truncated oligosaccharides, or mixtures of glycosylated and nonglycosylated monomers. 4 R. J. DeLange and T.-S. Huang, J. Biol. Chem. 246, 698 (1971). 5 In this chapter, a distinction is made between "nonglycosylation" and "deglycosylation"; in the former case the mechanism by which egg-white avidin has "lost" its oligosaccharide chains is unknown, wheras in the latter case the oligosaccharide residues of glycosylated forms of avidin are removed experimentally. Preliminary results using endoglycosidase F gave promising results in the deglycosylation of certain commercial preparations of avidin (e.g., Belovo) but not with others (e.g., avidin Ex, STC Laboratories Inc., Winnipeg, MB), suggesting qualitative differences in the oligosaccharide content of the two products (T. Viswanatha, personal communication). 6 In our laboratory, we have isolated from nature bacterial strains that are capable of producing deglycosylated avidin. We are currently in the process of isolating the enzyme(s) responsible for this activity.

METHODS IN ENZYMOLOGY, VOL. 184

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

[6]

NONGLYCOSYLATEDAVIDIN II

Applied

~(H2o)

"~ E

I

I

i

I

Buffer

i

69 [

I

I

I

aMM

1

o 2.c E 0

Cl

<0.5

I 0

200

250

i

300

I

350 Volume (ml) FIG. I. Preparative purification of nonglycosylated avidin by chromatography on Con Aagarose. A solution of Belovo avidin (-0.5 mg/ml in distilled water) was applied to a 25-ml Con A-agarose column until protein appeared in the effluent (thus indicating saturation of the column). The nonglycosylated avidin (shaded peak) was eluted by washing the column with Tris buffer. The glycosylated forms of avidin were eluted with three sequential volumes (-30 ml each) of the same buffer containing a-methyl-D-mannoside (aMM).

Affinity C h r o m a t o g r a p h y

Reagents Avidin 2 Con A - a g a r o s e (7-10 mg/ml resin, Sigma Chemical Co., St. Louis,

MO) Tris buffer (25 m M Tris-HCl, pH 7.4, containing 150 m M NaCI, 1 m M CaCI2, and I m M MgCI:) a - M e t h y l - D - m a n n o p y r a n o s i d e (5% aqueous solution)

Procedure Belovo avidin (87 mg) is dissolved in 192 ml distilled water, and the solution is centrifuged (10,000 g for 15 min). The clarified solution is applied to a Con A column (25 ml). Tris buffer is used to wash the column, after which the nonglycosylated avidin p e a k appears. Elution of the adsorbed glycosylated forms is accomplished with 5% a-methyl-D-mannopyranoside in the same buffer. The affinity chromatographic pattern of a representative preparation of nonglycosylated avidin is shown in Fig. 1. The fractions that contain nonglycosylated avidin (shaded area, Fig. 1) and those that are eluted

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.