ELSEVIER Journal of Biotechnology 46 ( 1996) 79-83

Biotechnology

Purification of peanut lectin using guar gum as an affinity ligand

Rem Tyagi, Ritu Agarwal, Munishwar N. Gupta *

Chemistry Department, Indian Institute of Technology, Haus Khas, New Delhi 110016. India

Received 29 September 1995; revised 27 November 1995; accepted 28 November 1995

Abstract

Affinity precipitation is a convenient purification approach which combines the advantages of precipitation with selectivity of affinity interactions. The main limitation of the technique at present is the cost of affinity ligands and matrix. In this work, peanut lectin was isolated by affinity precipitation using guar gum linked alginate. Both matrix as well as affinity ligand were inexpensive in nature. The recovered lectin (46 mg per 100 g seeds) purified by this single-step procedure appeared as a single band of M, 29000 on SDS-polyacrylamide gel electrophoresis.

Keywords: Affmity precipitation; Alginate; Galactose specific lectin; Peanut lectin; Guar gum; Protein purification

1. Introduction

Lectins have emerged as an important class of proteins having a wide variety of biochemical appli- cations (Goldstein and Poretz, 1986) including their use in bioseparation (Goldstein and Pore& 1986) and reversible immobilization (Cabral and Kennedy, 1993). One limitation in their large-scale use is possibly their high cost. Hence, it is worthwhile to develop efficient and economical processes for their purification. The existing methods invariably employ sugars and oligosaccharides as an affinity ligand in the final purification step in the multistep protocols (Goldstein and Poretz, 1986). These affinity ligands themselves are fairly expensive and contribute to the high purification costs. Naturally occurring polysac- charide material like guar gum are more economical substitutes. The present work shows that guru gum

l Corresponding author.

Ekevier Science B.V.

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works equally well as an affinity ligand for N-acetyl galactosamine specific lectin from peanuts. Further the macroligand may be attached to a reversibly soluble-insoluble and inexpensive polymer alginate and the conjugate may be used for affinity precipita- tion (Gupta and Mattiasson, 1994a, Gupta and Matti- asson, 1994b; Pets et al., 1991; Kumar and Gupta, 1994) of peanut lectin.

2. Material and methods

2.1. I. Materials

Peanuts were purchased from the local market and stored at 4°C until used. Guar gum was a gift from Dabur India Ltd., India. Alginate was purchased from Sigma Chemical Company, USA. Seralose 4B was from Sisco Research Laboratories, India. This agarose b&d matrix is similar to Sepharose 4B available from Pharmacia. All other reagents used were of analytical grade quality.

80 R. Tyagi et al./Journal of Biorechnology 46 (1996) 79-83

2.1.2. Preparation of peanut seed extract Peanut seed extract was prepared according to the

method of Pujol and Cesari (1986).

2.1.3. Acid treatment of guar gum solution Guar gum solution was prepared by dissolving 5

mg guar gum in 1 ml distilled water and centrifuged at 12000 X g for 20 mm to get a clear supematant. The acid treatment of guar gum was done by treating it with HCI (0.2 M) at 50°C for different time periods (1 h, 2 h, 3 h). The pH of the guar gum solution was adjusted to 7.0 after given time to stop the acid treatment.

2.1.4. Activation of Seralose 4B Epichlorohydrin activation of Seralose 4B was

carried out as described by Porath and Olin (1983). The activated resin was then equilibrated with phos- phate buffered saline (PBS; 0.15 M sodium phos- phate buffer, pH 7.2, containing 0.15 M NaCl).

2.15. Binding of guar gum/ acid treated guar gum samples with activated Seralose 4B

For binding, 15 ml of guar gum sample was added to 30 ml activated gel and incubated at 25°C for 2 h. The supematant was collected by centrifug- ing it at 12 000 X g for 15 min. Gel was then washed with PBS to further remove tmreacted guar gum. The extent of binding of guar gum to activated Seralose 4B was calculated by measuring guar gum concen- tration in supematant and washings using phenol sulphuric acid method (Chaplin, 1987).

2.1.6. Binding of peanut lectin to guar gum linked Seralose 4B resin

15 ml of peanut seed extract was added to 30 ml guar gum linked Seralose 4B and incubated at 25°C for 2 h in a water bath with continuous stirring. The supematant was collected by centrifugation at 12 000 x g for 15 min to remove unbound protein. The bound protein was then elukd with 0.05 M galactose (dissolved in PBS).

2.1.7. Activation of alginate 1 g alginate was dissolved in 20 ml distilled

water. About 0.5 ml epichlorohydrin and 5 mg sodium borohydride dissolved in 3 ml of 2 N NaOH were added to the alginate solution and stirred for 2 h at room temperature. An additional 1 ml of 2 N

NaOH and 0.5 ml of epichlorohydrin were added to the mixture and allowed to stir overnight on a mag- netic stirrer. For precipitation of activated alginate, the solution was diluted lOO-times and then 67 ml of 20 mM CaCl, was added to it. After 1 h incubation at 25”C, the precipitate was washed with 30 mM CaCl, six times (each time with 20 ml> to remove excess of reagents and finally dissolved in 20 ml of 0.1 M EDTA solution.

2.1.8. Binding of guar gum with activated alginate 20 ml of guar gum solution (2.5 mg ml-‘) was

added to 20 ml of activated alginate and the solution was stirred in water bath at 25°C for 2 h. The precipitation of alginate by CaCl, was done in the same way as described earlier. The precipitate thus obtained was washed with CaCl, solution to remove unbound guar gum and dissolved in 20 ml of 0.1 M EDTA solution. To calculate the extent of binding of guar gum to alginate the guar gum concentration was measured in the supematant and washings. For this purpose 2 ml of 1 M Na,SO, was added to 1 ml aliquot of each sample to precipitate the excess of Ca” (Limre et al., 1992) before the estimation of carbohydrate content by phenol sulphuric acid method (Chaplin, 1987).

2.1.9. Precipitation of peanut lectin using guar gum linked alginate

15 ml of peanut seed extract was dialyzed against 0.15 M NaCl, pH 7.0 and then added to 20 ml of guar gum linked alginate solution in 0.1 M EDTA and incubated at 25°C for 1 h in a water bath with continuous stirring. The alginate was then precipi- tated by adding 2 M CaCl, such that the final concentration of Ca2+ was 30 mM and washed with 30 mM CaCl, to remove unbound protein. The precipitate was again dissolved in 20 ml of 0.1 M EDTA solution and 5 ml of 0.2 M galactose solution was added to it to elute the bound protein. The alginate guar gum conjugate was then again precipi- tated as described before and centrifuged at 1200 X g for 20 min. The supematant was collected and dialyzed against phosphate buffered saline.

2.1 JO. Haemagglutinating activity The haemagglutinating activity of peanut lectin

was measured using glutaraldehyde treated

R. Tyagi et al./Journal of Biotechnology 46 (1996) 79-83 81

trypsinized rabbit blood cells (Turner and Liener, 1975).

2.1 .I 1. Electrophoresis Electrophoresis of peanut lectin sample under de-

natured conditions was carried out in 12% polyacryl- amide gel using Tris-glycine buffer, pH 8.3 (Hames, 1986).

2.1.12. Protein estimation Protein estimation was done according to the dye

binding method (Bradford, 1976).

3. Results and discussion

It was thought worthwhile exploring whether an affinity precipitation approach for galactose specific lectins may be developed based upon guar gum as an affinity ligand. Towards that aim, guar gum was first tried as the affinity ligand in the conventional affin- ity chromatography for purification of well charac- terized and galactose specific peanut lectin. It has been pointed out that sometime partially hydrolyzed polysaccharides chains bind better to peanut lectin (Pujol and Cesari, 1986). Hence, guar gum as such and acid treated samples for 1 h, 2 h and 3 h were linked to Seralose 4B activated with epichlorohydrin.

Out of 75 mg of guar gum as such and 75 mg of acid treated samples (1 h), 19.5 mg and 25.5 mg, respectively, were found to bind to 30 ml of epi- ahlorohydrin activited Seralose 4B. However, 2 h and 3 h acid treated guar gum samples did not show any binding to activated Seralose 4B. Therefore, for purification of peanut lectin, first two gels were used and it was found that when 15 ml of peanut seed extract (protein content - 117 mg) was added to 30 ml resin, the protein bound was found to be same in both the cases (Table 1). However, the yield of the lectin eluted from guar gum linked Seralose 4B by 0.05 M galactose was slightly more as compared to the lectin purified by acid treated guar gum linked Seralose 4B. Using higher concentrations of galac- tose (0.1 M/OS M) for elution does not affect the yield of the lectin. The protein eluted with 0.05 M galactose (in both the cases) was tested for lectin activity by haemagglutination assay. It has been shown earlier that by incorporating boric acid, this

Table 1

Purifcation of peanut lectin using guar gum linked Seralose 4B

Purification steps Protein (mg) % yield

1. Peanut seed extract 117 100

2. Procedure I (i) Protein bound to guar 74.8 64

gum linked Seralose 4B

(ii) Protein eluted with 0.79 0.7

0.05 M galactose

Procedure 2

(i) Protein bound to acid treated 76.5 65

guar gum linked Seralose 4B

(ii) Protein eluted with 0.05 M 0.62 0.5

galactose

Peanut seed (2.7 g) were suspended in 25 ml 0.015 M sodium

phosphate buffer pH 7.2, containing 0.15 M NaCI. homogenized

in a Waring blender and allowed to stand overnight at 4°C. After

centrifugation at 10000X g for 20 mitt, the lipid phase floating on

the top was discarded. The supematant (15 ml; 7.8 mg protein per

ml) was collected and used for further purification process. The

protein content in each step was measured by dye binding method.

test can be carried out with rabbit blood cells even in the presence of the specific sugar. The eluted protein in both the cases showed positive agglutination in the presence of borate (Tyagi and Gupta, 1993).

After having established guar gum as an effective affinity ligand, it was linked to alginate via epichlorohydrin activation method. Precipitation of alginate by Ca*+ is a well-known phenomenon (Smidsrod and Skjak-Break, 1990). Guar gum linked alginate was found to be quantitatively precipitated with Ca2+. In our protocol, to avoid an additional step of dialysis, guar gum linked alginate solution in 0.1 M EDTA was directly used for affinity precipita- tion. In this case also, guar gum linked alginate could be again precipitated quantitatively although a slightly higher concentration of Ca*+ was necessary for complete precipitation of the polymer. 30 mM CaCl, was found to completely precipitate 1% algi- nate guar gum conjugate solution (Fig. 1).

Peanut extract was added to the guar gum linked alginate solution in 0.1 M EDTA and after the incubation at 25°C it was precipitated with CaCl, as described in Materials and methods. About 63% of the total protein added is precipitated with alginate guar gum conjugate (Table 2). The lectin bound to

82 R. Tyagi et al. / Journal of Biotechnology 46 (1996) 79-83

Lc ok CoClz concentration (mtd)

Fig. 1. Precipitation of guar gum linked alginate by CaCl,. ( 8 )

Guar gum linked alginate, (0) guar gum linked algiuate in the

presence of 0.1 M EDTA.

conjugate was specifically eluted with 0.05 M galac- tose. Due to the presence of both CaCl, and EDTA, activity could not be detected by incorporating bo- rate and protein had to be dialyzed extensively against 0.15 M NaCl before its activity was checked. It showed positive agglutination against trypsinized rabbit blood cells. For the control the same amount of peanut extract and alginate solution were taken and precipitation with CaCl, was done. About 20% of the protein added is precipitated, however, noth- ing is specifically eluted with 0.05 M/0.1 M galac- tose.

The homogeneity of the lectin was checked on SDS-PAGE and it was found to move as a single band of IV, 29000 (Fig. 2). This agrees well with the reported molecular weight (M, 27 000 - 28 000) of the subunit lectin (Goldstein and Poretz, 1986).

It may be relevant to compare this affinity precipi- tation protocol with the purification procedures re- ported by others. Pujol and Cesari (1986) have briefly

Table 2 Precipitation of peanut lectin using alginate guar gum conjugate

Purification steps

1. Peanut seed extract

Protein (mg) % yield

117 100 2. Protein precipitated with algiuate-guar 73.9 63 gum conjugate

3. Protein recovered with 0.05 M 1.23 1.1 galactose

7he details of preparing peanut seed extract are same as given in the legend to Table 1. In this case also, The isolation was started

with 2.7 g peanut seeds and initial crude extract was 15 ml (7.8

mg protein per ml).

1 M kDa

Fig. 2. SDS-PAGE gel. Lane 1: purified peanut lectin; lane M:

marker proteins.

reviewed the existing data and report that yields vary from 40 mg to 75 mg of lectin from 100 g of peanut seeds. Most of the procedures have used more than one step and, in some cases, the purified lectin has been found to be electrophoretically heterogenous. The best yields have been reported by Lotan et al. (1975) viz. 65-75 mg lectin per 100 g peanut seeds, but the affinity chromatography matrix (Sepharose- epsilon-aminocaproyl-P_Dgalactopyranoside) used is rather expensive. The affinity precipitation, a single- step procedure reported here yields about 46 mg electrophoretically homogenous lectin per 100 g peanut seeds. It is also likely that the lectin content may vary with specific strain of seeds used. Most of the workers, including ourselves, have used seeds bought from the local market.

Thus, an affinity conjugate consisting of alginate and guar gum, both inexpensive polysaccharides, was useful for precipitation of peanut lectin which is a galactose specific lectin. It is likely that the method described here would work for all the galactose specific lectins (e.g., Jacalin, Sunhemp lectin, Castor bean lectin). Galactose specific lectins constitute an important class of lectins (Goldstein and Pore& 1986). Also, as discussed elsewhere (Gupta and Mat- tiasson, 1994a, Gupta and Mattiasson, 1994b), affn- ity precipitation methods, like the one described here, are easy to scale up and may be employed directly with crude homogenates.

R. Tyagi et al./Jownal of Biotechnology 46 (1996) 79-83 83

Acknowledgements

This work was supported by the Department of Science and Technology (Govt. of India) in the form of a Young Scientist project grant to RT, Council of Scientific and Industrial Research in the form of SRF to RA and research grants to MNG from Department of Biotechnology (Govt. of India) and Swedish agency for research cooperation with developing countries (SAREC).

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