Separation of insect hemolymph proteins by cascade-mode multiaffinity chromatography.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 20:243-251 (1992) Separation of Insect Hemolymph Proteins by Cascade-Mode MuItiaff inity Chromatography Preminda Samaraweera, JerkerPorath, and John H. Law Centerfor Insect Science (P.S., J.H.L.) and Depurtment of Biochemistry (P.S., J.P., J.H.L.), University of Arizona, Tucson The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multiaffinity chromatography (CASMAC) on a main-line tandem column chain containing Zn2+-TED, T-gel, Ni2+-DPA, and phenylsepharose and a side-line column containing Zn2+-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph. @ 1992 Wiley-Llss, Inc. Key wards: Manduca sexta, metal ion affinity, multiaffinity adsorption INTRODUCTION Using conventional methods, it has been possible to isolate and characterize every major protein of the hemolymph of the adult hawkmoth, Manducu sexta [l].There are, however, many additional proteins that can be seen in 2-D" gels of this fluid. If we are to gain knowledge of the various minor proteins, many of which may have vital functions, we must resort to new methodologies. While in theory it should be possible to isolate sufficient quantities of the minor proteins from 2-D gels for N-terminal sequence, and perhaps for production of antibodies, both of which should facilitate cloning and sequencing, in practice this is frequently difficult or impossible. The task would be made easier if one could first fractionate hemolymph to yield less complex Acknowled ments: This work was supported by the grant GM29238 from the National Institutes of He3th. Received December 23,1991; accepted April 14,1992. Address reprint requests to Dr. John H. Law, De artment of Biochemistry, Biological Sciences West Building, Universityof Arizona, Tucson, d85721. *Abbreviations used: CASMAC = cascade mode multiaffinity chromatography; 2-D = two dimensional; D = Dalton; DPA = dipicolylamine agarose; IMA = immobilizedmetal ion affinity; IMAC = immobilized metal ion affinity chromatography; SDS = sodium dodecyl sulfate; TED = tris-carboxymethylethylene diamine agarose; T-gel = thiophilic gel. 0 1992 Wiley-Liss, Inc. 244 Sarnaraweera et al. mixtures, and then use simple gel electrophoresis for the final isolation. This strategy also yields proteins in the native, rather than denatured, state. Among the newer technologies that might be useful for this purpose are IMAC and the combination of IMAC with other affinity methods, for example, CASMAC . IMAC has been demonstrated to be a useful separation method for a variety of proteins [4-61, and CASMAC has been successfully applied for the separation of components of human serum . We recently reported the use of a ferric ion IMAC method for a one-step isolation of insect vitellins . We will now report the use of CASMAC to separate proteins of adult hemolymph of Manduca sexta. The method yields several fractions of relatively simple composition, and some of the known major proteins have been identified in these fractions. In addition, some previously undescribed proteins have been revealed by this separation technique. MATERIALS AND METHODS Insects M . sextu eggs were supplied by Drs. J.P. Reinecke and J.S. Buckner, U.S. Department of Agriculture, Fargo, ND. Animals were raised as previously described . Antibodies to M . sextutd Hemolymph Proteins Rabbit polyclonal antibodies against apolipophorin I11 [ 101, microvitellogenin [ll],transferrin , ommochrome-binding protein , and vitellogenin  were prepared in this laboratory. Anti-serpin antibodies were kindly provided by Dr. Michael Kanost of Department of Biochemistry, the University of Arizona. Hemolymph Collection and Density Gradient Centrifugation Adult M . sextu (40-50; male or female)were bled by the "flushing out" method of Chino et al.  using 0.1 M sodium phosphate, 0.15 M NaCl, pH 7, that contained 10 mM diisopropylfluorophosphate and 50 mM glutathione for injection into the moths. The hemolymph preparation was then subjected to KBr density gradient centrifugation  at 50,000 rpm in a Beckman VTi 50 rotor at 5°C for 16 h, and the lipoprotein-free subnatant was collected. The preparation was then dialyzed against 60 mM sodium phosphate, pH 7.5. Chromatography Media Phenyl-sepharose and butyl-sepharose were from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Preparation of DPA, TED, and T-gel have been described [7,17,18]. DPA and TED, respectively, bound 45 and 58 pmol Cu2+ ions/ml adsorbent. Immobilization of metal ions (Ni2+or Zn2') was performed by passing 50 ml of 20 mM metal ion solution through the column containing DPA or TED (8-10 ml of gel). The column was then washed with distilled water followed by starting buffer. Zn2+-DPA and Zn2+-TEDwere reloaded with metal ions after each use because possible migration and leakage of the metal ion in the bed cannot be followed by visual inspection. Ni2+-DPAwas used without any reloading. Hemolymph Proteins of Manduca sexfa 245 Chromatography All chromatography runs were performed at room temperature in 1.5 cm x 10 cm columns (Bio-Rad Laboratories, Richmond, CA)at a flow rate of 1mb'min, In order to determine the affinities of different proteins for different adsorbents preliminary experiments were conducted usin individual columns. The bed volumes were as follows: Ni2+-DPA,8 ml; Zn'+-DPA, 8 ml; Zn2+-TED, 10 ml; butyl-sepharose, 14 ml; phenyl-sepharose, 14 ml; and T-gel, 15ml. Each column was equilibrated with 50 mM sodium phosphate, pH 7.6, that contained 0.6 M K2S04 (buffer I). "Antichaotropic" sulfate was added to a high concentration in order to suppress simple ionic interaction and to intensify coordinate binding. Two milliliters of the lipophorin-free hemolymph prepared as above was brought into the same buffer by adding solid K2S04, dilution with water and pH adjustment, and applied to each column. The optical absorbance at 280 nm of the hemolymph preparation was 5-6 absorbance units. Therefore the total optical absorbance of the material used in all chromatographic runs amounted to 10-12 units. After washing the columns with buffer I to remove unbound and loosely bound proteins, the columns containing Ni2+-DPA,Zn2+-DPA, and &?+-TED were developed with 50 mM sodium phosphate, 0.6 M K2S04,0.1 M imidazole, pH 7.6 (buffer 11), followed by 0.5.M sodium acetate, 0.5 M NaC1, pH 5.5 (buffer 111) (compared to sulfate, acetate and chloride are located in the direction of increasing chaotropic behavior on the Hofmeister scale). Those containing butyl-sepharose, phenylsepharose, and T-gel were successively eluted with buffers of decreasing polarity, namely, 50 mM sodium phosphate, pH 7.6 (buffer IV), 40% ethylene glycol in buffer IV (buffer V), and 30% 2-propanol in buffer IV (buffer VI). The elution of proteins from the columns was monitored by measuring the optical absorbance at 280 nm. The proteins eluted on each development step were analyzed by SDS-PAGE. On the basis of the preliminary investigations columns containing Zn2+-TED, T-gel, Ni2+-DPA, and phenyl-sepharose were connected in series to give a main-line tandem column (Fig. 1). The tandem column equilibrated in buffer I was loaded with the hemolymph preparation (brought into buffer I as above) and unbound and loosely bound proteins were washed out. This initial washing was routinely carried out with 180 ml of buffer I; all the other chromatography steps used 60 ml of the respective buffers. In all cases, 3 ml fractions were collected. The non-interacting fraction was denoted fraction A. Individual columns were then disconnected and developed separately. The column containing Zn2+-TEDwas successively developed with buffers I1 and I11 to give fractions B and C, respectively. T-gel was washed with buffer IV and the effluent was directed to a column containing Zn2+-DPA. The fraction which was eluted without any interaction with Zn2+-DPAwas denoted D. The two columns were then disconnected, and T-gel was eluted with buffer V followed by buffer VI to give fractions E and F. Zn2'-DPA was washed with buffers I1 and 111. These two fractions were denoted G and H, res ectively. Ni2+-DPAwas developed in the same way as Zn2'-TED and Zn -DPA, i.e., with buffers I1 and 111, to give fractions I and J. Phenylsepharose was successively eluted with buffers IV, V, and VI to obtain fractions K, L, and M. r+ 246 Samaraweera et ill. NiZf-DPA 1 A Fig. 1. Schematic drawing of the arrangement of the tandem columns. The main-line consisting of Zn+’-TED, 1-Gel, Ni’+-DPA, and phenyl-sepharose (Phe.Seph.) is shown on the left side of the diagram. 4 indicates elution of columns, and 3 indicates disconnection of the bed. The Roman numerals I-VI represent buffer systems used to develop the columns, and A-M indicate the fractions obtained. For example, the 1- el column was disconnected from the tandem column, and eluted with buffer IV into the Zn’+-DPA column. The material which did not bind to either column was designated fraction D. The 1-gel column was again disconnected, and eluted with buffers V and VI to give fractions E and F, respectively. SDS-Polyacrylamide Gel Electrophoresis SDS-polyacrylarnide slab gel electrophoresis was carried out as described by Laemmli 1191. The proteins were routinely analyzed in 4-15% gradient gels under reducing conditions. Immunoblotting The proteins on SDS-polyacrylamide gels were slectrophoretically blotted onto nitrocellulose membranes [ZO]in a Hoefer Scientific Instruments TE-52 Hernolymph Proteinsof Manduca sexta 247 transphor electrophoresis unit (San Francisco, CA). Immunoblotting was per formed using rabbit anti-protein antibodies and Vectastain ABC reagent kit (Vector Laboratories Inc. Burlingame, CA) as recommended by the manufacturer. RESULTS Affinities of Munducu Hernolymph Proteins for Different Adsorbents The initial studies using individual columns were performed to determine adsorption behavior of hemolymph proteins toward the different adsorbents. Both quantitative and qualitative differences in adsorptions were observed between different gels. Under the experimental conditions used, phenylsepharose and Ni2+-DPAbound more than 80% of applied hernolymph proteins, whereas T-gel, butyl-sepharose, and %'+-TED adsorbed some 20-30%, Zn2+-DPAadsorbed about 60% of the hernolymph proteins. Adsorption of Hernolymph Proteins by Tandem Column The different adsorbents in the tandem column (Fig. I) were arranged depending on their affinities for hemolymph proteins. Since Zn2+-TED adsorbed the least amount of protein this gel was placed first in the tandem column. The T-gel adsorbed some additional proteins, and therebre, was placed next to Zn2"-TED column. The Ni2+-DPAcolumn was inserted next and phenyl-sepharose which adsorbed highest amount of protein was placed Iast. Figure 2 shows a typical composite elution profile of the chromatography of adult female Munducu hemolymph as described in the Materials and Methods section. The amounts of proteins recovered in the fractions (as the percentage of applied proteins) are summarized in Table 1. The majority of the proteins was bound to Ni2+-DPAand eluted in fractions I and J. Zn'+-TED bound (fractionsB and C) only 10%of the applied proteins. This value contrasts with the finding using the individual column, where it bound about 20% of the applied proteins. Although it is unlikely, the reduction of binding of proteins by Zn2+-TED in the tandem column may be due to extended washing involved in the procedure. The results of SDS-PAGE of eluted fractions are shown in Figure 3. The intensities of the protein bands in Figure 3 are not indicative of the proportion of the proteins in the original hemolymph preparation: in order to make the bands visible in the Coomassie blue stained gel, certain fractions (E and F, for example) required loading of as much as 60-70% of the preparation, while some (fractions I and J) yielded sufficiently intense bands with less than 5% of the fraction*Some of the protein bands present in SDSpolyacrylamide gel were identified by immunoblotting, and are indicated in the same figure. Only a few proteins were present in fraction A, and microvitellogenin was the major constituent. Fraction B contains a number of proteins, but, as can be seen when compared to the applied hemolymph preparation (Fig. 3, lane He), almost all of them seem to be minor constituents. Similarly, fractions D-H contained mainly minor hemolymph proteins. Transfenin and vitellogenin are the major proteins in fraction I while traces of insecticyanin are also present. More than 90% of insecticyanin together with some vitellogenin and ommochrome-binding protein were eluted in fraction J. Serpin is a Sarnaraweera et al. 1 0.30 iJ 0.20 0.10 0.00 0 80 4 0 160 120 Fraction 200 240 280 Number Fig. 2. The composite elution profile obtained on fractionation of adult female Manduca hemolymph by CASMAC. Arrows with I-VI indicate starting elution positions with respective buffers. A-M represent peaks corresponding to each fraction. constituent in fraction K, and apolipophorin I11 was eluted in fraction L in a relatively pure form. While vitellogenin was observed in many fractions the amounts present in fractions E, G, and H represent only a small percentage. The majority of the protein is present in fractions I and J. The distribution of vitellogenin in different fractions may be due to its heterogeneity in terms of phosphorylation and glycosylation. An unidentified protein with approximate M, 30 kD is eluted in fraction M in a considerably purified form. Again, this protein seems to represent a less abundant constituent in Munducu hemolymph. The results of the fractionation of male hemolymph are very similar to that of female hemolymph except that peak J is higher than peak I in the chromatogram, and vitellogenin subunits and microvitellogenin bands are absent in SDS-polyacrylamide gel for male hemolymph (not shown). TABLE 1. Recoveries of Proteins in the Fractions Obtained From CASMAC (Expressedas the % of applied sample) Fraction A % recovery 4 B 7 C 1 D 9 2 E 2 F 2 G 2 H 2 5 I 1 J 8 K 1 L 1 3 M 5 Hernolymph Proteins of Manduca sexfa 249 Fig. 3. SDS-PAGE of the fractions collected on CASMAC. The analysiswas performed on 4-15% gradient gels under reducingconditions. Lanes: Mo, molecularweight markers; He, hernolymph subnatant after density gradient centrifugation; A-M, materials in fractions A-M, respectively. Proteins identified by irnmunoblotting are indicated with arrows. a, vitellogenin large subunit; b, transferrin; c, vitellogenin small subunit; d, rnicrovitellogenin; e, insecticyanin; f , apolipophorin I l l ; g, ornrnochrorne-bindingprotein; h, serpin. DISCUSSION Although a considerable body of knowledge has been accumulated about Munducu hemolymph proteins [l],most of the past studies were focused on the major components. The fact that the less abundant proteins have drawn little attention can be mainly attributed to the difficulty of purification which is a prerequisite for their characterization and understanding of their biological role. The present investigation was undertaken to explore the possibility that CASMAC will yield less complex mixtures of hemolymph proteins and facilitate purification of minor proteins. To achieve this objective we utilized IMA gels combined with thiophilic and hydrophobic gels in a manner described by Porath and Hansen . Since we lack an understanding of the interaction of M. sextu hemolymph proteins with different affinity adsorbents, the choice of the adsorbents and the order in which they were arranged in the tandem column were empirical. With the exception of vitellogenin, we were able to separate many of the known adult Munducu hemolymph proteins from each other. For example, the procedure resolved microvitellogenin, transferrin, insecticyanin, and apolipophorin I11 into different fractions (fractions A, I, J, and L, respectively). The distribution of vitellogenin in more than one fraction may reflect heterogeneity in its composition. We have previously reported a simple one-step IMAC purification of vitellogenin .The recovery of the 250 Samaraweera et id. CASMAC was high; we recovered more than 90% of the proteins (in terms of absorbance at 280 nm) applied to the tandem column chain. This less than 100% recovery may be due to insertion of T-gel and phenyl-sepharose in the tandem column; during the initial tests using individual adsorbents the IMA gels yielded close to 100% recovery while with T-gel and phenyl-sepharose only 85-90% of the proteins could be recovered. We were also able to separate by CASMAC some less abundant proteins into simple mixtures. Repetition of the experiment a number of times yielded the same results. Currently we are attempting to purify minor proteins from these fractions. Immobilized metal ion affinity gels are prepared by coupling a ligand to an insoluble matrix, and coordinating metal ions to the ligand. Although IMAC is a rather new technique it may have wide applications in separation of a variety of biomolecules because one can combine a large number of ligands and a large number of metal ions to obtain an enormous number of affinity adsorbents. Variations in binding and elution of proteins are possible by changing the experimental conditions (ionic strength, pH, etc.). Here we used one hydrophobic adsorbent, one thiophilic adsorbent, and three kinds of IMA gels. Further resolution of hemolymph proteins should be possible by inserting more IMA gels and other types of adsorbents (ion exchange gels, for example) into the main-line tandem or as side-line columns. The separation can be further improved by introducing salt or pH gradients to displace bound proteins. LITERATURE CITED 1. Kanost MR, Kawooya JK, Law JH, Ryan RO, Van Heusden MC, Ziegler R Insect haemolymph proteins. Adv Insect Physiol22,299 (1990). 2. Porath J, Carlsson J, Olsson I, Belfrage G: Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258,598 (1975). 3. Porath J: Salting-outadsorption techniques for protein purification. Biopolymers 26(Suppl.), S193 (1987). 4. Muszynska G, Anderson L, Porath J: Selective adsorption of phosphoproteins on gel- immobilized ferric chelate. Biochemistry 25,6850 (1986). 5. Nakagawa Y, Yip T, Belew M, Porath J: High-performance immobilized metal ion affinity chromatography of peptides: Analytical separation of biologically active synthetic peptides. Anal Biochem 268,75 (1988). 6. Mantovaara T, Pertoft H, Porath J: Purification of factor VI1I:c coagulant activity from rat liver nonparenchymal cell culture medium by immobilized metal ion affinity chromatography. Biotechnol Appl Biochem 13,120 (1991). 7. Porath J, Hansen P: Cascade-mode multiaffinity chromatography-fractionation of human serum proteins. J Chromatogr 550,751 (1990). 8. Van Heusden MC, Fogarty S, Porath J, Law JH: Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate. Protein Expression and Purification 2, 24 (1991). 9. Prasad SV, Ryan RO, Law JH, Wells MA: Changes in lipoprotein composition during larvalpupal metamorphosis of an insect, Manducu sextu. J Biol Chem 262,558 (1986). Hernolyrnph Proteinsof Manduca sexta 251 10. Kawooya JK, Keim PS, Ryan RO, Shapiro JP,Samaraweera I?, Law JH: Insect apolipophorin 111. Purification and properties. J Biol Chem 259,10733 (1984). 11. Kawooya JK, Osir EO, Law JH: Physical, chemical and immunologicalproperties of microvitellogenin. J Biol Chem 262,10844 (1986). 12. Bartkld NS, Law JH: Isolation and molecular cloning of transferrin from the tobacco hornworm, Munduca sextu. J Biol Chem 265,21684 (1990). 13. Martel RR, Law JH: Purification and properties of an ommochrome-binding protein from the hemolymph of the tobacco hornworm, Munducu sextu. J Biol Chem 266,21392 (1991). 14. 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