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Purification and Characterization of Bacillus thuringiensis var. tenebrionis Insecticidal Proteins produced in E. coli S. C. Macintosh, S. L. McPhersonr,
F. J. Perlak, P. G. Marronez and R. L. Fuchs
Monsanto Agricultural Company, A Unit of Monsanto Company, 700 Chesterfield Village Parkway, Saint Louis, Missouri 63 198 Received
June
11,
1990
Native and single amino acid variants of the Bacillus thuringiensisvar. tenebrionis insecticidal proteins were expressedin Escherichiacoli, purified andexaminedfor biological and biochemical properties. A novel, pH dependent,preferential precipitation method was implementedto purify Escherichiacoli producedBacillus thuringiensisvar. tenebrionis proteins, which are active against Coloradopotato beetle {Leptinotarsadecemlineata)larvae. Cysteineresiduesof the native Bacillus thuringiensisvar. tenebrionisprotein werereplacedby serineresiduesby site-directedmutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionisproteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteineresidue(aminoacid 540) causedcompleteinactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidalactivities comparableto eachother andto solubilizedcrystalsfrom the original strain. 01990Academic Press,Inc. Bacillus thuringiensis(B.t. )is a gram-positive, sporeforming bacteriumthat characteristicallyproducesa parasporalcrystal protein which exhibits insecticidalactivity. NumerousB.t. strainshave beenidentified that are active againstlepidopterans,dipteransand coleopteraninsects(1). Strain classificationby flagellar serotypeandcrystal structuregenerally reflects insect specificity (2). Analysis of B.t. protein structureasit relatesto activity hasonly recently beeninvestigated with mostof the studiescenteredon the lepidopteranactive proteins, suchas thosefrom B.t. var. kurstaki (B.t.k.) and B.t. var. thuringiensis (3,4). The parasporalbipyramidal crystals from these lepidopteran-activestrains consistof largeprotoxins with molecularweights between130to 140 kDa containing 13to 17 cysteineresidues($6). Once proteolyzed in the insectmidgut, the molecularweight is reducedto 63 to 70 kDa and all the cysteineresiduesareeliminated(56). Although studiesthat characterizethe disulfide bond structureof theseB.t. full length crystal proteinshave beenlargely inconclusive,this high cysteinecontent may indicate that the disulfide
1 Centerfor AIDS Research,UAB, Birmingham,Alabama, 35294. 2 Entotech, Inc., 1497Drew Ave., Davis, California, 95616. Abbreviations: B.t., Bacillus thuringiensis,B.t.k., Bacillus thuringiensis var. kurstaki, B.t,t., Bacillus thuringiensisvar.tenebrionis, DTNB, 5,5’-dithiobis(2-nit-benzoic acid); EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; TNB 2-, 2-nitro-5-thiobenzoicacid; RB, refractile bodiesor inclusion bodies. 0006-291X/90
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bonds are required to form and maintain crystal structure. Most reports (7,8,9) used indirect techniques to study the disulfide bonds in B.r.k. crystals. Solubilization of B.r.k. crystals requires an alkaline environment with reducing agents such as dithiothreitol or thioglycollate (7). This implies that disulfide bonds must be cleaved to achieve solubilization. These physiological conditions mimic that of the lepidopteran larval midgut, which exhibits a reducing enviornment of high alkaline pH (10 to 11.5) (10). Inhibition of crystal solubilization and insecticidal activity by chemically blocking the thiol groups supports the importance of the disulfide bond(s) (9). Detailed studies on the structure of the dipteran-active B.f. var. isruelensis crystal proteins (11) are difficult to interpret since these strains contain multiple crystal types composed of a wide variety of proteins. Couche et al. (11) identified both intra- and inter-chain disulfide bonds in solubilized whole crystal preparations and located these disulfide bonds in at least three distinct B.t. var. israelends proteins. In contrast to B.t.k. (7), chemical modification of thiol groups of the mosquitocidal B.t. var. israelensis crystal proteins had little effect on toxicity (12). Similar data are not yet available for the more recently isolated B.t. var. renebrionis (B.t.t.) crystal proteins (13,14,15). It is unlikely that the protein subunits within the crystal are linked by disulfides since pH 10 buffers alone, without reducing agents, completely dissolves B.t.t. crystal protein(s) (16). Our group has cloned and sequenced the gene which encodes the full length B.t.t. insecticidal protein and related truncated proteins (17). In this study, we describe the isolation of these proteins in a highly purified form. The deduced amino acid sequence of B.t.t. established that the protein contains three cysteine residues. Each of the three cysteine residues was individually modified to a serine by site-specific mutagenesis to determine whether the protein contained disulfide bonds and to establish the importance of these residues for insecticidal activity. In addition, a B.t.t. gene was constructed that encodes a protein with all three cysteine residues modified to serine residues. The modified proteins were purified and the free cysteine content was determined.
Finally, the insecticidal activity of truncated and amino acid variants of B.t.t. were
compared to native BL?. proteins. MATERIALS
AND
METHODS
Reagents. Guanidine HCl was obtained from Schwarz/Mann Biotech (Cleveland, OH) 5,5’-dithiobis(2-nitbenzoic acid) (DTNB) and all other chemicals were reagent grade and purchased from Sigma Chemical Co. (St, Louis, MO). All electrophoresis chemicals were obtained from Bio-Rad Laboratories (Richmond, CA). Bacterial strains and culture conditions. The B.t.t. strain, obtained from W. Schnetter (13) was grown and crystals isolated and solubilized as described (17). All genes encoding the B.t.r. proteins and amino acid variants (Table 1) were cloned behind the Escherichia coli recA promotor and bacteriophage ‘I7 gene10 translational enhancer (18). Abbreviations for the amino acid variants are also listed in Table 1. Cultures of E. coli JMlOl containing the appropriate B.t.t. plasmids were grown and harvested as described previously (17). Site-directed mutagenesis of cysteine residues. The procedure of Kunkle (19) was used to mutagenize the truncated B.r.t. band 3 protein gene. Synthetic oligonucleotides (3@ mers) were used to change the cysteine residues to serine. The fiit cysteine to serine change at amino acid 243, was tgt to tct (pMON5345); the second at amino acid 478, tgc to age (pMON5338); and the third at amino acid 540 was tgc to tcg (pMON5339). Simultaneous alteration of all three cysteine residues to serines were also done by site-directed mutagenesis (pMON5384). All three mutations were sequenced in each plasmid and in all cases the protein of the expected size was detected by western analysis (20) (data not shown). Purification of B&t. proteins produced in E. coli. Cell pellets derived from the E. coli JMlOl strains containing the appropriate plasmids (Table 1) were suspended in water to a
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TABLE
B.r.2. proteins produced
1,2,3,4
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Characteristics of Bacillus thuringiensis var. tenebrionis proteins produced from designatedplasmids Plasmid
_------
Host
Modified Amino acid number
Abbreviation
B.t.t.
---
--- - __-
1
pMON5460
E. coli
486
bandI
193 3 ,4
pMON5436
E. coli
_-_
band 1+3
pMON5456 pMON5469
E. coli E. coli
band 3 band4
3 3
pMON5345
E. coli E. coli
49 57d 243e 478~
C478S
3
pMON5338 pMON5339
3
pMON5384
Insecticidal activitya
C243S
E. coli
54oe
c54os
E. coli
243.478,540=
C243,478,5408
QInsect bioassays were performed against Colorado potato beetle larvae. b Thr changed to Asp. c Met changed to Ile. d N-terminal 57 amino acids deleted, Met added before amino acid 58. e Cys changed to Ser.
final concentrationequalto l/20 of the fermentationvolume. The suspended cells were lysed by sonicationin an ice bath with a Heat SystemsUltrasonicssonicator(Lakeview, New York) at a power of 9,50% duty cycle for a total of 5 minutes. The sonicatedpreparationwascentrifuged for 20 minutesat 20,000 X g at 4’C. Pellets,containing refractile bodiesand cell debris,were washed twice with cold water, once with 2M sodiumchloride andfinally, suspended in one-third the original volume of 100mM sodiumcarbonate,pH 10. After stirring at room temperaturefor 2 hours,the solution was centrifuged for 20 minutesat 20,000X g at 4°C to remove insoluble material. Supematantprotein from the truncated and singleaminoacid variants (B.t.r. band 3, band 1+3, band4 andC243S and C478S)were concentratedat 4°C on a YM-10 membraneusingan Amicon stirred cell concentrator(Danvers,MA) to a final protein concentrationof 5 to 10 mg per ml. The concentratedprotein preparationwasdialyzed extensively (48 hr) against10 mM sodium phosphate,pH 6.0 buffer, which preferentially precipitated the B.r.r. protein. The precipitated protein wasremoved by centrifugation at 20,000X g for 20 minutesat 4’C anddissolvedin one-fifth the original volume of 100mM sodiumcarbonate,pH 10 or pH 11 buffer. B.r.t. band 1 protein was purified by first dialyzing the dilute, solubilizedprotein preparationagainst10 mM sodiumphosphate,pH 6.0 buffer at 4’C, then concentrating IO-fold in anAmicon stirredcell concentrator,conditionswhich precipitatedthe band 1 protein. After centrifuging for 20 minutesat 20,000 X g at 4OC,the pellet wasdissolvedin 100mM sodium carbonate,pH 10 and the B.t.t. band 1 protein wasfurther purified using a Superose12 FPLC sizing column (PharmaciaAB, Uppsala,Sweden). Purification of B.t.t. modified cysteine variants from E. coli JMlOl. While the proteinsfrom the C243Sand C478Svariants were purified to near homogeneityby the differential purification methoddescribedabove,the other cysteinevariants, C54OSand C243,478,54OSwere not solubilized from refractile bodiesusingalkaline pH buffers. Instead, 4M guanidineHCl wasrequiredto solubilize theseproteins. The sulfhydryl content was not determinedsincethe purity of thesetwo variants waslessthan 50%. E. coli extracts containing theseamino acid variants were concentrated10X and comparedwith E. coli extracts of B.t.t. band 3 for insecticidal activity. 667
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Protein determination. Protein concentrations were routinely determined by the method of Bradford (21), using bovine serum albumin as the standard. Protein concentrations for experiments measuring sulfhydryl residues were determined using a theoretical extinction coefficient of El%=15 cm-1 calculated from the known amino acid sequence. Sulfhydryl content. The concentration of sulfhydryl groups was determined by titration using DTNB as described by Ellman (22). Typically, B.t.t. protein samples (80 L) containing 0.4 to 1.O mg protein were mixed with 880 h of deoxygenated buffer; 100 mM sodium phosphate, 1 mM EDTA, 6.4 M guanidine HCl, pH 7.3 and incubated at room temperature for 10 minutes. DTNB (40 h) was added to a final concentration of 0.2 mM and the solution was gently mixed to avoid oxidation. After an additional 15 minute incubation period the absorbance at 412nm of the solutions were measured. The same experiment was repeated under reducing conditions by overnight dialysis of the same proteins in the deoxygenated buffer described above with the addition of 10 mM dithiothreitol (DTT). The DIT was subsquently removed by passing the protein solutions through two sequential PD-10 columns (Pharmacia AB) equilibrated with the initial deoxygenated buffer without DTT. Samples of the dialysate buffer, as a control, were processed in the same manner. Immediately after elution from the second column, the protein and buffer samples were titrated with DTNB as described above, except the volumes of protein and buffer were altered to reflect the dilution effect of the gel filtration treatment. An extinction coefficient of 13,600 M-1 cm-1 (22) was used to quantitate the titration product, 2-nitro5-thiobenzoic acid (TNB 2-). Data represent the average of three experiments. Insecticidal activity. Insecticidal activity of the various B.t.t. culture and protein preparations was assayed employing newly hatched Colorado potato beetle (Leptinmzrsa decemheafa) larvae in the tomato leaf feeding assay described previously (17). Tween-20 was used at a final concentration of 0.3%. to facilitate adherence of the solution to the leaf surface. RESULTS
AND DISCUSSION
Four distinct proteins were identified from B.t.t. crystal preparations by SDS-PAGE analysis (23) (Fig. 1). For convenience, these proteins were labelled B.t.t. band 1 through 4 proteins in order of decreasing molecular mass (Fig. 1). We have shown previously (17) that all of these crystal proteins are derived from a single gene through the utilization of two distinct initiation codons and proteolytic cleavage. Differential solubilities of the various B.r.t. proteins and amino acid variants compared to contaminating proteins was exploited to develop rapid and effective purification protocols. B.t.r. proteins corresponding to band 1, band 1+3, band 3, band
B.t.t.
Figure 1. Identification of B.r.t. proteins produced by B.t.t. and by E. coli JMlOl plasmid containing strains. The B.t.r. proteins were purified as described in Materials & Methods and separated on a 9% SDS-PAGE gel that was silver stained (14). Four distinct B.I. var. cenebrionis proteins were labelled band 1 through 4 in order of decreasing molecular weight as indicated.
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Table 2. Purification
Step
of Bacillus
Total protein (mg)
Sonicated Refractilecells bodies SolubilizedRBs pH precipitation
12soo 1725 644
310
AND
BIOPHYSICAL
thuringiensis
Total B.r.r.0 protein (mg)
897 875 543 298
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var. tenebrionis from E. coli
B.2.t. protein/ total protein
0.52 0.07 0.84 0.96
Yield (%I
:t 62 34
0BIT. protein,JMlOl pMON 5456,wasestimated by densitometric scanning of SDS-PAGEgels.
4, C243Sand C478Swere purified asdescribedin Materials and Methods. All six proteins preferentially and efficiently precipitatedanddissolvedat pH 10 with the exception of band 4. B.t.t. band 4 failed to completely dissolveuntil the pH wasraisedto 11. All proteinspurified by this pH precipitation method,were greaterthan 95% pure asjudged by densitometricscanningof SDS-PAGE gels(Fig. 1) and analytical chromatographyon Superose12 (datanot shown). Recoveriesof 35%, basedupon densitometicscans,were observedduring a typical purification (Table 2, Fig. 2). Data in Figure 2 summarizethe relative yields and purity of the B.t.t. band3 protein at the various stagesof this novel purification procedure. A numericalsummary(Table 2) wasderived basedupon independentquantitationof protein concentrationsanddensitometic scanningof SDS-PAGE gelsto estimatethe concentrationof B.t.t. band 3 protein. One to two milligramsof pure B.t.t. protein was typically obtainedper gramof E. coli cell wet weight. Quantitiesof up to 300 mg of highly pure B.t.t. band3 protein wasobtainedfrom the cells derived from a singleten liter fermentation. Four molar guanidineHCl wasrequiredto solubilize protein correspondingto C54OSand C243,478,54OS from inclusion bodies. Unfortunately this harshtreatment alsosolubilized a numberof contaminants(data not shown)andrenderedthe protein insecticidally inactive (Table 1). Efforts to purify theseB.t.t. variant proteins,which included gel filtration, ion exchangeand immunoaffinity chromatography,were unsuccessful.The guanidineHCl solubilizedvariants elutedfrom a gel filtration columnin an aggregatedform with an estimatedmolecularmassof approximately 1,000kDa (data not shown). The purification difficulties encounteredwith these two variants suggestthat the protein folded improperly when aminoacid 540 wasmodified. Another site-directedmutant wasconstructed(S. McPherson,unpublished)that removed four amino acidsfrom the C-terminus. This protein was alsoshownto be insecticidally inactive. These data suggestthat the C-terminal sequenceof B.t.t. protein may be extremely important in determiningthe confirmational structureof the protein andthereforethe volubility and insecticidal activity. DTNB titration, to estimatesulfhydryl content, wasnot performedon the folded, native proteins due to the insolubility of the B.t.t. protein(s) at neutral PH. TNB titration of the unfolded, non-reducedproteinsin the presenceof guanidineHCl maintainedprotein solubility. These analysisidentified 2.6 cysteineresiduesfor eachband 3 protein molecule(MW = 67 kDa). The
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Figure 2. Analysis of B.r.t. band 3 protein purification. Equivalent amounts of B.t.t. band 3 on a 9%SDS-PAGEthatwassilverstained protein from each purification step were separated (14). sulfhydryl content of the reduced,unfolded band3 protein was 2.5 cysteineresiduesper molecule, which is similarto the expectedvalue of 3 cysteineresiduesfrom the deducedamino acid sequence (17). DTNB titration of the two singleaminoacid variants, designatedC243S and C478S,under non-reducedconditionsrevealed 1.4 and 1.6 cysteine residues,respectively. Both variants under reducedconditions showed1.9 cysteineresiduesper molecule. Becausea singlecysteine was modifed in eachprotein the predictedvalue was2 cysteineresidues.There wasno difference in thiol titration betweenreducedand non-reducedforms of any of theseproteins. The DTNB titration dataestablishedthe lack of an intramoleculardisulfide bond andalso suggeststhat no intermoleculardisulfide bondsexist. The only possibleconformation of the B.f.t. protein that might contain an intermoleculardisulfide bond, basedon thesedata, would link C540 betweentwo B.t.t. molecules. The sulfhydryl content of the two variants, C54OSand C243,478,54OS, was not estimatedsincepurified protein could not be obtained. Despitethe inability to estimatethe sulfhydryl content of C54OSor C243,478,54OS this theory is improbablesinceit would predict 3 cysteineresiduesfor the reducedB.t.t. band 3 protein, but only 2 cysteinesfor the non-reduced molecule. Likewise, both C243Sand C478S,would have 2 cysteine residuesfor the reducedand only one residuefor the non-reduced.Data above showedno significant difference in the titrated cysteineswith the reducedand non-reducedproteins. Furthermore, sincehigh pH buffer in the absenceof reducing agentscompletely dissolvesB.t.t. protein, intermoleculardisulfide bondsare unlikely. Insecticidal activities of all the purified proteinswere comparedto the solubilizedB.t.t. proteins (bands1,2,3, and 4) obtained from B.t.f. crystals. Relative activities from all purified protein preparationswere comparableto one anotherand to the solubilizedB.t.t. crystal proteins (Table 1). No synergistic activity wasobservedwith either full length and truncatedB.u. proteins 670
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or with the combination of B.r.r. proteins solubilized from B.r.r. crystals.
These data further support the lack of structural disulfides for the native B.r.r. protein since single amino acid variants C243S and C478S showed similar biological activities. Comparison of the insecticidal proteins from B.r.r. and B.r.k. show interesting similarities and differences.
Although the full-length B.r.k. protoxins contain multiple cysteine residues linked
by disulfide bonds (7), the insecticidally active protease resistant toxins do not (7,ll) eliminating the role of disullide bonds for insecticidal activity. Disulfide bonds in the full length proteins may be required for appropriate protein folding, for determining the tertiary structure, crystal formation and perhaps crystal stability in the B.r.k. strain. Yet, protein folding and crystal formation in the B.r.r. strain is not a function of disulfide bonds based on our data. However the crystal shape does differ from that found in B.r.k. (1). The insecticidally active proteins from both B.r.k. and B.r.r. are similar in size (-63 to 73 kDa) and are devoid of disulfide bonds. Both proteins have been expressed in plants at levels that confer insect tolerance (24,25, Fischhoff, unpublished). Therefore disuifide bonds, which typically maintain tertiary protein structure and function, are not required for B.r.r. or B.r.k. insecticidal activity when produced in the heterologous bacterium, E. coli, or in plants.
Acknowledgments:
We thank Bruce Bishop for fermentation support. We are also grateful to
David Fischhoff and Steve Padgette for support of this project and critical review of this manuscript.
REFERENCES
1: t. 5. 6. 7. Glb. 11. 12. 13. 14.
Hiifte, H., and Whiteley, H.R. (1989) Microbial. Rev. 53, 242-255. Aronson, AI., Beckman, W., and Dunn, P. (1968) Microbial. Rev. 50, l-24. Huber, H.E., Liithy, P., Ebersold, H. and Cordier, J. (1981) Arch. Microbial. 129, 14-18. Hofte, H., VanRie, J., Jansens, S., VanHoutven, A., Vanderbruggen, H., and Vaeck, M. (1988) Appl. Envior. Microbial. 54,2010-2017. Fischhoff, D.A., Bowdish, K.S., Perlak, F.J., Marrone, P.G., McCormick, S.M., Niedermeyer, J.G., Dean, D.A., Kusano-Kretzmer, K., Mayer, E.J., Rochester, D.E., Rogers, S.G. and Fraley, R.T. (1987) Bio-Technology, 5,815-917. Adang, M. J., Staver, M. J., Rocheleau, T.A., Leighton, J., Barker, R.F., and Thompson, D.V. (1985) Gene 36,289-300. Nickerson, K.W. (1980) Biorech. Bioeng. 23, 1305-1333. Tojo, A. (1986) Agric. Biol. Chem. 50, 157-162. Bulla Jr., L.A., Kramer, K.J., and Davidson, L.I. (1977) J. Bacrer. 130, 375-383. Wolfson, J.L., Murdock, L.L. (1990) J. Chem. Ecol. 16, 1089-1102. Couche, G.A., Pfannenstiel, M.A., and Nickerson, K.W. (1987) J. Bacrer. 169,3281-3288. Pfannenstiel, M.A., Couche, G.A., Muthukumar, M., and Nickerson, K.W. (1985) Appl. Envior. Microbial. 50, 1196-l 199. Krieg, A., Huger, A.M., Langerbrook, G.A. and Schnetter, W. (1983) Parhoryp. 2. Ang. Enr. 96, 500-508. &5y3;;dt, C. Soares, G.G., Wilcox, E.R. and Edwards, D.L. (1986) BiolTechnology 4,
15. Krieg, A., Huger, A.M., Langerbruch, G.A. and Schnetter, W. (1984) Ang. Schadlingskde., Pjlanzenschurz, Unwelrschurz. 57, 145-150. 16. Bernhard, K. (1986) FEMS Microbial. L&r. 33,261-265. 17. McPherson, S.A., Perlak, F.J., Fuchs, R.L., Marrone, P.G., Lavrik, P.B., and Fischhoff, D.A. (1988) Biolrechnology 6,61-66. 18. Olins, P.O., Rangwala, S.H. (1990) Methods in Enzymology 185, 115-l 19. 671
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19. Kunkle, T.A. (1985) Proc. Natl. Acad. Sci. 82, 488-492. 20. Towbin, H.T., Staelhelin, T. and Gordon, J. (1979) Proc. Natl. Aced. Sci. USA 76, 43504354. 21. Bradford, M.M. (1976) Anal. Biochem. 72,248-254. 22. Ellman, G.L. (1959) Arch. Biochem. Biophy. 82,70-77. 23. Laemmli, U.K. (1970) Nature 227,681-685. 24. Von Montagu, M.C.E., Vaeck, M.A., Zabeau, M.F.O., Leemans, J.J.A. andHofte, H.F.P. (1986) Modifying plants by genetic engineering to combat or control insects. European Patent Application 8630091.1 25. Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Anal. Biochem. 118, 197-203.
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Purification and Characterization of Bacillus thuringiensis var. tenebrionis Insecticidal Proteins produced in E. coli S. C. Macintosh, S. L. McPhersonr,
F. J. Perlak, P. G. Marronez and R. L. Fuchs
Monsanto Agricultural Company, A Unit of Monsanto Company, 700 Chesterfield Village Parkway, Saint Louis, Missouri 63 198 Received
June
11,
1990
Native and single amino acid variants of the Bacillus thuringiensisvar. tenebrionis insecticidal proteins were expressedin Escherichiacoli, purified andexaminedfor biological and biochemical properties. A novel, pH dependent,preferential precipitation method was implementedto purify Escherichiacoli producedBacillus thuringiensisvar. tenebrionis proteins, which are active against Coloradopotato beetle {Leptinotarsadecemlineata)larvae. Cysteineresiduesof the native Bacillus thuringiensisvar. tenebrionisprotein werereplacedby serineresiduesby site-directedmutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionisproteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteineresidue(aminoacid 540) causedcompleteinactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidalactivities comparableto eachother andto solubilizedcrystalsfrom the original strain. 01990Academic Press,Inc. Bacillus thuringiensis(B.t. )is a gram-positive, sporeforming bacteriumthat characteristicallyproducesa parasporalcrystal protein which exhibits insecticidalactivity. NumerousB.t. strainshave beenidentified that are active againstlepidopterans,dipteransand coleopteraninsects(1). Strain classificationby flagellar serotypeandcrystal structuregenerally reflects insect specificity (2). Analysis of B.t. protein structureasit relatesto activity hasonly recently beeninvestigated with mostof the studiescenteredon the lepidopteranactive proteins, suchas thosefrom B.t. var. kurstaki (B.t.k.) and B.t. var. thuringiensis (3,4). The parasporalbipyramidal crystals from these lepidopteran-activestrains consistof largeprotoxins with molecularweights between130to 140 kDa containing 13to 17 cysteineresidues($6). Once proteolyzed in the insectmidgut, the molecularweight is reducedto 63 to 70 kDa and all the cysteineresiduesareeliminated(56). Although studiesthat characterizethe disulfide bond structureof theseB.t. full length crystal proteinshave beenlargely inconclusive,this high cysteinecontent may indicate that the disulfide
1 Centerfor AIDS Research,UAB, Birmingham,Alabama, 35294. 2 Entotech, Inc., 1497Drew Ave., Davis, California, 95616. Abbreviations: B.t., Bacillus thuringiensis,B.t.k., Bacillus thuringiensis var. kurstaki, B.t,t., Bacillus thuringiensisvar.tenebrionis, DTNB, 5,5’-dithiobis(2-nit-benzoic acid); EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; TNB 2-, 2-nitro-5-thiobenzoicacid; RB, refractile bodiesor inclusion bodies. 0006-291X/90
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bonds are required to form and maintain crystal structure. Most reports (7,8,9) used indirect techniques to study the disulfide bonds in B.r.k. crystals. Solubilization of B.r.k. crystals requires an alkaline environment with reducing agents such as dithiothreitol or thioglycollate (7). This implies that disulfide bonds must be cleaved to achieve solubilization. These physiological conditions mimic that of the lepidopteran larval midgut, which exhibits a reducing enviornment of high alkaline pH (10 to 11.5) (10). Inhibition of crystal solubilization and insecticidal activity by chemically blocking the thiol groups supports the importance of the disulfide bond(s) (9). Detailed studies on the structure of the dipteran-active B.f. var. isruelensis crystal proteins (11) are difficult to interpret since these strains contain multiple crystal types composed of a wide variety of proteins. Couche et al. (11) identified both intra- and inter-chain disulfide bonds in solubilized whole crystal preparations and located these disulfide bonds in at least three distinct B.t. var. israelends proteins. In contrast to B.t.k. (7), chemical modification of thiol groups of the mosquitocidal B.t. var. israelensis crystal proteins had little effect on toxicity (12). Similar data are not yet available for the more recently isolated B.t. var. renebrionis (B.t.t.) crystal proteins (13,14,15). It is unlikely that the protein subunits within the crystal are linked by disulfides since pH 10 buffers alone, without reducing agents, completely dissolves B.t.t. crystal protein(s) (16). Our group has cloned and sequenced the gene which encodes the full length B.t.t. insecticidal protein and related truncated proteins (17). In this study, we describe the isolation of these proteins in a highly purified form. The deduced amino acid sequence of B.t.t. established that the protein contains three cysteine residues. Each of the three cysteine residues was individually modified to a serine by site-specific mutagenesis to determine whether the protein contained disulfide bonds and to establish the importance of these residues for insecticidal activity. In addition, a B.t.t. gene was constructed that encodes a protein with all three cysteine residues modified to serine residues. The modified proteins were purified and the free cysteine content was determined.
Finally, the insecticidal activity of truncated and amino acid variants of B.t.t. were
compared to native BL?. proteins. MATERIALS
AND
METHODS
Reagents. Guanidine HCl was obtained from Schwarz/Mann Biotech (Cleveland, OH) 5,5’-dithiobis(2-nitbenzoic acid) (DTNB) and all other chemicals were reagent grade and purchased from Sigma Chemical Co. (St, Louis, MO). All electrophoresis chemicals were obtained from Bio-Rad Laboratories (Richmond, CA). Bacterial strains and culture conditions. The B.t.t. strain, obtained from W. Schnetter (13) was grown and crystals isolated and solubilized as described (17). All genes encoding the B.t.r. proteins and amino acid variants (Table 1) were cloned behind the Escherichia coli recA promotor and bacteriophage ‘I7 gene10 translational enhancer (18). Abbreviations for the amino acid variants are also listed in Table 1. Cultures of E. coli JMlOl containing the appropriate B.t.t. plasmids were grown and harvested as described previously (17). Site-directed mutagenesis of cysteine residues. The procedure of Kunkle (19) was used to mutagenize the truncated B.r.t. band 3 protein gene. Synthetic oligonucleotides (3@ mers) were used to change the cysteine residues to serine. The fiit cysteine to serine change at amino acid 243, was tgt to tct (pMON5345); the second at amino acid 478, tgc to age (pMON5338); and the third at amino acid 540 was tgc to tcg (pMON5339). Simultaneous alteration of all three cysteine residues to serines were also done by site-directed mutagenesis (pMON5384). All three mutations were sequenced in each plasmid and in all cases the protein of the expected size was detected by western analysis (20) (data not shown). Purification of B&t. proteins produced in E. coli. Cell pellets derived from the E. coli JMlOl strains containing the appropriate plasmids (Table 1) were suspended in water to a
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TABLE
B.r.2. proteins produced
1,2,3,4
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Characteristics of Bacillus thuringiensis var. tenebrionis proteins produced from designatedplasmids Plasmid
_------
Host
Modified Amino acid number
Abbreviation
B.t.t.
---
--- - __-
1
pMON5460
E. coli
486
bandI
193 3 ,4
pMON5436
E. coli
_-_
band 1+3
pMON5456 pMON5469
E. coli E. coli
band 3 band4
3 3
pMON5345
E. coli E. coli
49 57d 243e 478~
C478S
3
pMON5338 pMON5339
3
pMON5384
Insecticidal activitya
C243S
E. coli
54oe
c54os
E. coli
243.478,540=
C243,478,5408
QInsect bioassays were performed against Colorado potato beetle larvae. b Thr changed to Asp. c Met changed to Ile. d N-terminal 57 amino acids deleted, Met added before amino acid 58. e Cys changed to Ser.
final concentrationequalto l/20 of the fermentationvolume. The suspended cells were lysed by sonicationin an ice bath with a Heat SystemsUltrasonicssonicator(Lakeview, New York) at a power of 9,50% duty cycle for a total of 5 minutes. The sonicatedpreparationwascentrifuged for 20 minutesat 20,000 X g at 4’C. Pellets,containing refractile bodiesand cell debris,were washed twice with cold water, once with 2M sodiumchloride andfinally, suspended in one-third the original volume of 100mM sodiumcarbonate,pH 10. After stirring at room temperaturefor 2 hours,the solution was centrifuged for 20 minutesat 20,000X g at 4°C to remove insoluble material. Supematantprotein from the truncated and singleaminoacid variants (B.t.r. band 3, band 1+3, band4 andC243S and C478S)were concentratedat 4°C on a YM-10 membraneusingan Amicon stirred cell concentrator(Danvers,MA) to a final protein concentrationof 5 to 10 mg per ml. The concentratedprotein preparationwasdialyzed extensively (48 hr) against10 mM sodium phosphate,pH 6.0 buffer, which preferentially precipitated the B.r.r. protein. The precipitated protein wasremoved by centrifugation at 20,000X g for 20 minutesat 4’C anddissolvedin one-fifth the original volume of 100mM sodiumcarbonate,pH 10 or pH 11 buffer. B.r.t. band 1 protein was purified by first dialyzing the dilute, solubilizedprotein preparationagainst10 mM sodiumphosphate,pH 6.0 buffer at 4’C, then concentrating IO-fold in anAmicon stirredcell concentrator,conditionswhich precipitatedthe band 1 protein. After centrifuging for 20 minutesat 20,000 X g at 4OC,the pellet wasdissolvedin 100mM sodium carbonate,pH 10 and the B.t.t. band 1 protein wasfurther purified using a Superose12 FPLC sizing column (PharmaciaAB, Uppsala,Sweden). Purification of B.t.t. modified cysteine variants from E. coli JMlOl. While the proteinsfrom the C243Sand C478Svariants were purified to near homogeneityby the differential purification methoddescribedabove,the other cysteinevariants, C54OSand C243,478,54OSwere not solubilized from refractile bodiesusingalkaline pH buffers. Instead, 4M guanidineHCl wasrequiredto solubilize theseproteins. The sulfhydryl content was not determinedsincethe purity of thesetwo variants waslessthan 50%. E. coli extracts containing theseamino acid variants were concentrated10X and comparedwith E. coli extracts of B.t.t. band 3 for insecticidal activity. 667
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Protein determination. Protein concentrations were routinely determined by the method of Bradford (21), using bovine serum albumin as the standard. Protein concentrations for experiments measuring sulfhydryl residues were determined using a theoretical extinction coefficient of El%=15 cm-1 calculated from the known amino acid sequence. Sulfhydryl content. The concentration of sulfhydryl groups was determined by titration using DTNB as described by Ellman (22). Typically, B.t.t. protein samples (80 L) containing 0.4 to 1.O mg protein were mixed with 880 h of deoxygenated buffer; 100 mM sodium phosphate, 1 mM EDTA, 6.4 M guanidine HCl, pH 7.3 and incubated at room temperature for 10 minutes. DTNB (40 h) was added to a final concentration of 0.2 mM and the solution was gently mixed to avoid oxidation. After an additional 15 minute incubation period the absorbance at 412nm of the solutions were measured. The same experiment was repeated under reducing conditions by overnight dialysis of the same proteins in the deoxygenated buffer described above with the addition of 10 mM dithiothreitol (DTT). The DIT was subsquently removed by passing the protein solutions through two sequential PD-10 columns (Pharmacia AB) equilibrated with the initial deoxygenated buffer without DTT. Samples of the dialysate buffer, as a control, were processed in the same manner. Immediately after elution from the second column, the protein and buffer samples were titrated with DTNB as described above, except the volumes of protein and buffer were altered to reflect the dilution effect of the gel filtration treatment. An extinction coefficient of 13,600 M-1 cm-1 (22) was used to quantitate the titration product, 2-nitro5-thiobenzoic acid (TNB 2-). Data represent the average of three experiments. Insecticidal activity. Insecticidal activity of the various B.t.t. culture and protein preparations was assayed employing newly hatched Colorado potato beetle (Leptinmzrsa decemheafa) larvae in the tomato leaf feeding assay described previously (17). Tween-20 was used at a final concentration of 0.3%. to facilitate adherence of the solution to the leaf surface. RESULTS
AND DISCUSSION
Four distinct proteins were identified from B.t.t. crystal preparations by SDS-PAGE analysis (23) (Fig. 1). For convenience, these proteins were labelled B.t.t. band 1 through 4 proteins in order of decreasing molecular mass (Fig. 1). We have shown previously (17) that all of these crystal proteins are derived from a single gene through the utilization of two distinct initiation codons and proteolytic cleavage. Differential solubilities of the various B.r.t. proteins and amino acid variants compared to contaminating proteins was exploited to develop rapid and effective purification protocols. B.t.r. proteins corresponding to band 1, band 1+3, band 3, band
B.t.t.
Figure 1. Identification of B.r.t. proteins produced by B.t.t. and by E. coli JMlOl plasmid containing strains. The B.t.r. proteins were purified as described in Materials & Methods and separated on a 9% SDS-PAGE gel that was silver stained (14). Four distinct B.I. var. cenebrionis proteins were labelled band 1 through 4 in order of decreasing molecular weight as indicated.
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Table 2. Purification
Step
of Bacillus
Total protein (mg)
Sonicated Refractilecells bodies SolubilizedRBs pH precipitation
12soo 1725 644
310
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Total B.r.r.0 protein (mg)
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var. tenebrionis from E. coli
B.2.t. protein/ total protein
0.52 0.07 0.84 0.96
Yield (%I
:t 62 34
0BIT. protein,JMlOl pMON 5456,wasestimated by densitometric scanning of SDS-PAGEgels.
4, C243Sand C478Swere purified asdescribedin Materials and Methods. All six proteins preferentially and efficiently precipitatedanddissolvedat pH 10 with the exception of band 4. B.t.t. band 4 failed to completely dissolveuntil the pH wasraisedto 11. All proteinspurified by this pH precipitation method,were greaterthan 95% pure asjudged by densitometricscanningof SDS-PAGE gels(Fig. 1) and analytical chromatographyon Superose12 (datanot shown). Recoveriesof 35%, basedupon densitometicscans,were observedduring a typical purification (Table 2, Fig. 2). Data in Figure 2 summarizethe relative yields and purity of the B.t.t. band3 protein at the various stagesof this novel purification procedure. A numericalsummary(Table 2) wasderived basedupon independentquantitationof protein concentrationsanddensitometic scanningof SDS-PAGE gelsto estimatethe concentrationof B.t.t. band 3 protein. One to two milligramsof pure B.t.t. protein was typically obtainedper gramof E. coli cell wet weight. Quantitiesof up to 300 mg of highly pure B.t.t. band3 protein wasobtainedfrom the cells derived from a singleten liter fermentation. Four molar guanidineHCl wasrequiredto solubilize protein correspondingto C54OSand C243,478,54OS from inclusion bodies. Unfortunately this harshtreatment alsosolubilized a numberof contaminants(data not shown)andrenderedthe protein insecticidally inactive (Table 1). Efforts to purify theseB.t.t. variant proteins,which included gel filtration, ion exchangeand immunoaffinity chromatography,were unsuccessful.The guanidineHCl solubilizedvariants elutedfrom a gel filtration columnin an aggregatedform with an estimatedmolecularmassof approximately 1,000kDa (data not shown). The purification difficulties encounteredwith these two variants suggestthat the protein folded improperly when aminoacid 540 wasmodified. Another site-directedmutant wasconstructed(S. McPherson,unpublished)that removed four amino acidsfrom the C-terminus. This protein was alsoshownto be insecticidally inactive. These data suggestthat the C-terminal sequenceof B.t.t. protein may be extremely important in determiningthe confirmational structureof the protein andthereforethe volubility and insecticidal activity. DTNB titration, to estimatesulfhydryl content, wasnot performedon the folded, native proteins due to the insolubility of the B.t.t. protein(s) at neutral PH. TNB titration of the unfolded, non-reducedproteinsin the presenceof guanidineHCl maintainedprotein solubility. These analysisidentified 2.6 cysteineresiduesfor eachband 3 protein molecule(MW = 67 kDa). The
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Figure 2. Analysis of B.r.t. band 3 protein purification. Equivalent amounts of B.t.t. band 3 on a 9%SDS-PAGEthatwassilverstained protein from each purification step were separated (14). sulfhydryl content of the reduced,unfolded band3 protein was 2.5 cysteineresiduesper molecule, which is similarto the expectedvalue of 3 cysteineresiduesfrom the deducedamino acid sequence (17). DTNB titration of the two singleaminoacid variants, designatedC243S and C478S,under non-reducedconditionsrevealed 1.4 and 1.6 cysteine residues,respectively. Both variants under reducedconditions showed1.9 cysteineresiduesper molecule. Becausea singlecysteine was modifed in eachprotein the predictedvalue was2 cysteineresidues.There wasno difference in thiol titration betweenreducedand non-reducedforms of any of theseproteins. The DTNB titration dataestablishedthe lack of an intramoleculardisulfide bond andalso suggeststhat no intermoleculardisulfide bondsexist. The only possibleconformation of the B.f.t. protein that might contain an intermoleculardisulfide bond, basedon thesedata, would link C540 betweentwo B.t.t. molecules. The sulfhydryl content of the two variants, C54OSand C243,478,54OS, was not estimatedsincepurified protein could not be obtained. Despitethe inability to estimatethe sulfhydryl content of C54OSor C243,478,54OS this theory is improbablesinceit would predict 3 cysteineresiduesfor the reducedB.t.t. band 3 protein, but only 2 cysteinesfor the non-reduced molecule. Likewise, both C243Sand C478S,would have 2 cysteine residuesfor the reducedand only one residuefor the non-reduced.Data above showedno significant difference in the titrated cysteineswith the reducedand non-reducedproteins. Furthermore, sincehigh pH buffer in the absenceof reducing agentscompletely dissolvesB.t.t. protein, intermoleculardisulfide bondsare unlikely. Insecticidal activities of all the purified proteinswere comparedto the solubilizedB.t.t. proteins (bands1,2,3, and 4) obtained from B.t.f. crystals. Relative activities from all purified protein preparationswere comparableto one anotherand to the solubilizedB.t.t. crystal proteins (Table 1). No synergistic activity wasobservedwith either full length and truncatedB.u. proteins 670
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or with the combination of B.r.r. proteins solubilized from B.r.r. crystals.
These data further support the lack of structural disulfides for the native B.r.r. protein since single amino acid variants C243S and C478S showed similar biological activities. Comparison of the insecticidal proteins from B.r.r. and B.r.k. show interesting similarities and differences.
Although the full-length B.r.k. protoxins contain multiple cysteine residues linked
by disulfide bonds (7), the insecticidally active protease resistant toxins do not (7,ll) eliminating the role of disullide bonds for insecticidal activity. Disulfide bonds in the full length proteins may be required for appropriate protein folding, for determining the tertiary structure, crystal formation and perhaps crystal stability in the B.r.k. strain. Yet, protein folding and crystal formation in the B.r.r. strain is not a function of disulfide bonds based on our data. However the crystal shape does differ from that found in B.r.k. (1). The insecticidally active proteins from both B.r.k. and B.r.r. are similar in size (-63 to 73 kDa) and are devoid of disulfide bonds. Both proteins have been expressed in plants at levels that confer insect tolerance (24,25, Fischhoff, unpublished). Therefore disuifide bonds, which typically maintain tertiary protein structure and function, are not required for B.r.r. or B.r.k. insecticidal activity when produced in the heterologous bacterium, E. coli, or in plants.
Acknowledgments:
We thank Bruce Bishop for fermentation support. We are also grateful to
David Fischhoff and Steve Padgette for support of this project and critical review of this manuscript.
REFERENCES
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