Analytical Chemistry of Bacillus thuringiensis

Chapter 8

Downloaded by UNIV OF PITTSBURGH on July 7, 2014 | http://pubs.acs.org Publication Date: July 10, 1990 | doi: 10.1021/bk-1990-0432.ch008

Development of a H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y A s s a y for Bacillus thuringiensis v a r . san diego δ-Endotoxin

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Leonard Wittwer , Denise Colburn , Leslie A. Hickle , and T. G. Sambandan 2

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Invitrogen Corporation, 1158 Sorrento Valley Road, Suite 20, San Diego, CA 92121 Mycogen Corporation, 5451 Oberlin Drive, San Diego, CA 92121

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A Reverse Phase HPLC assay has been developed for quantifying the delta -endotoxin produced by Bacillus thuringiensis var. san diego. The effects of ethyleneglycol as a mobile phase modifier and in stabilizing solubilized delta -endotoxinare discussed. Bacillus thuringiensis var. san diego (BTSD) is a naturally occurring bacillus which w isolated at Mycogen Corporation. It produces a protein delta-endotoxin as a crystalline inclusion body which is active against certain coleopteran insects such as Colorado Potato Beetle and Elm Leaf Beetle (1.2). This protein is the active ingredient in M-One® insecticide. An analytical assay is needed to quantify the native toxin levels in both fermentation batches and final product formulations. Currently the toxin is measured by SDS-PAGE (3) followed by laser densitometry. Further, to confirm the assay, a bioassay is also performed. This technique, although very sensitive, is not selective. SDS-PAGE measures total protein of a given molecular weight and does not differentiate between the active and the non-active forms of the endotoxin protein. As such, the assay has been difficult to correlate with the Bioassay (4). Furthermore, the assays were cumbersome, time-consuming, and the results can vary widely ( ±10% Standard Deviation). Hence, there was a need for a faster and more accurate assay to measure the endotoxin. Reverse Phase HPLC has gained popularity as a tool for quantitation and purity analysis of proteins. The mechanism of separation on the micro hydrocarbon bonded silica particulates is essentially hydrophobic interaction. This technique can separate proteins of similar molecular weights and other hydrophobic proteins. The purpose of this investigation was to develop a stability-indicating high performance liquid chromatography (HPLC) method for the quantitation of endotoxin in both the broth and in the final product. Materials and Methods Preparation of Endotoxin. Standard toxin used was made by isolation and recrystalization from BTSD grown in production media. The fermentation broth was concentrated three-fold either by centrifugation or ultrafiltration. The pH of the concentrate was raised to 12 -12.5 with 1 Ν NaOH. The mixture was stirred at room temperature for 20 minutes to dissolve the 0097-6156/90/0432-0070$06.00/Ό © 1990 American Chemical Society In Analytical Chemistry of Bacillus thuringiensis; Hickle, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

8.

WITTWERET AL.

An HPLC Assay for a δ-Endotoxin

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toxin. Insoluble material was removed by œntrifugation at 3500 RPM for 20 minutes. The supernatant was dialyzed against Tris- (50 mM Tris pH 7.5,1 mM EDTA) for 2 hours at 4°C. As the pH of the sample dropped, the toxin precipitated inside the dialysis tube. The precipitated toxin was collected and washed with water until the resulting pellet was white and the supernatant was clear.


On SDS-PAGE the crystals yield a single band with an "apparent" molecular weight of about 64 kD. The crystalized toxin standard is stable on SDS, Page, as well as by bioassay for at least six months when stored in 10 mM Tris-HCl buffer pH 7.5 at 4°C. Sample Preparation for HPLC. The toxin standard was prepared by dissolving the toxin crystals in 10 mM NaOH and 1 mM Dithioerythritol (DTT). Ethylene glycol was added to 50% U/V and incubated at 100°C for 10 minutes. The protein content was determined by a Lowry assay using BSA as the standard. It should be noted that the determination of M-One protein by using BSA as standards would contribute to a systematic error. We chose to neglect it for its contribution was small compared to other experimental errors. HPLC Equipment and Reagents. A Water's gradient system was used (680 gradient controller, Two - 510 pumps, U6K injector, 410 (UV) flow Detector). Data was collected with Gilson's 712 software package on a Dell System 310 computer. The column used was an Alltech Macrospere C4 (25CM 5 um particle size, 300 A pore). Detection was set at 254 nM and solvent flow was 1 ml/mm. Mobile phases A and Β were water and acetonitrile (ACN) respectively. Both eluants were modified with ethylene glycol and Trifluoroacetic acid (TFA) as described in the text. All reagents were HPLC grade. Results and Discussion Preparation of endotoxin solution from the crystal suspension was difficult. The crystal did not dissolve easily in mild solvents. The endotoxin dissolved in mobile phase (40% water 60% acetonitrile 0.1% TFA) would form a gel after a few minutes at room temperature. Crystals were also difficult to dissolve in urea or guanidine. Further alkali solubilized toxin was unstable. However, a solution of stable endotoxin was prepared by adding an equal volume of modified sample mix (2% SDS 10% Glycerol 2.5% Mercaptoethanol) to alkali solubilized endotoxin and heating the mixture at 100°C for 10 minutes. The resulting solution was stable and was suitable for HPLC analysis. As with most protein analyses, a gradient HPLC Method was developed to analyze the M-One standard. Preliminary experiments with mobile phases also indicated that TFA was essential for the elution of endotoxin. Although endotoxin gave the two characteristic peaks, ghost peaks were also encountered. For example, in the experiment when the standard was run with the following conditions isocratic 40% Β for 10 minutes and a gradient to 70% Β for 30 minutes and a return to 40% Β in 5 minutes, a third peak appeared during the return to initial conditions. The peak was analyzed by SDS-PAGE and was found to be 64 kD endotoxin. To minimize ghosting and to enhance recovery, addition of ethylene glycol to the mobile phase was implemented. As shown in Figure 1, an addition of 2% ethylene glycol to the mobile phase eliminated the late peak and ghost peak in the blank runs. The two peaks of endotoxin raised questions of purity of the product. Are two different proteins present or do the proteins exist as two forms (active and the non-active form)? To answer these questions, the various peaks were collected and were run on SD-PAGE. Both the peaks showed identical bank patterns to that of M-One, suggesting that the endotoxin existed in two forms. To study the various forms, a thermal study was undertaken. The experiment was conducted on a sample that was made in 50% glycerol. As shown in Figure 2, thefirstpeak was

In Analytical Chemistry of Bacillus thuringiensis; Hickle, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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ANALYTICAL CHEMISTRY OF BACILLUS THURINGIENSIS

Figure 1 A:

Effect of Ethylene Glycol in the Mobile Phase Mobile Phase: Gradient: Sample:

Figure IB:

A - Water, 0.015% TFA Β - Acetonitrile, 0.015% TFA 10 minutes at 45% B, gradient to 90% Β in 30 minutes BTSD crystals, dissolved in NaOH, SDS

Effect of Ethylene Glycol in the Mobile Phase Mobile Phase: Gradient: Sample:

A - Water, 2% ethylene glycol, 0.015% TFA B- Acetonitrile, 2% ethylene glycol, 0.015% TFA 10 minutes at 40% B, gradient to 70% Β in 30 minutes BTSD crystals, dissolved in NaOH, SDS



Figure 2:

No Heat 40

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50

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80°

Gradient: Sample:

Mobile Phase:

100

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A - Water, 2% ethylene glycol, 0.1 % TFA Β - Acetonitrile, 2% ethylene glycol, 0.1% TFA 10 minutes at 40% B, gradient to 70% Β in 39 minutes BTSD crystals, dissolved in NaOH, SDS, 50% glycerol heated 15 minutes

Effect of Heating the Delta-Endotoxin Sample in Glycerol

30"

VJ


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essentially being converted to the second peak by just heating the sample. In fact, only one form, the second peak, was observed when the sample was heated to 100°C, indicating the second peak is a denatured form of endotoxin. Clearly, this assay could be used as a stabilityindicating assay. Preliminary studies with bioassay indicate that the native protein was indeed active and the others had minimal activity. The native protein could also be of different molecular weight.


During the course of this investigation, it was also discovered that the denatured protein could be renatured by addition of ethylene gjycol. As shown in Figure 3, using the same experiment except substituting ethylene glycol for 50% glycerol, the sample returns to the native state on heating at 100°C. We have no explanation for this behavior. Further work is underway to study the mechanism of renaturation. Various dilutions of the toxin standard were injected to produce a calibration curve based on peak area (Figure 4). Multiple 50 ul injections of the same protein concentration yielded peak areas with a coefficient of variation 0.05%. Toxin Quantitation Samples of formulated BTSD toxin (M-One, Mycogen Corporation) were assayed by comparison to the calibration curve. The formulation was diluted with water and centrifuged to recover the toxin crystals. Chromatograms of this material yielded both peaks characteristic of the standard toxin injections. No combination of heat, ethylene glycol SDS, or glycerol converted the formulated toxin to one peak. Rather than assume the peak areas were additive, the quantitation scheme shown in Figure 5 was employed to measure toxin levels. A comparison of toxin values obtained from HPLC and SDS-PAGE assays are presented in Table I.

Table I.

Comparison of HPLC and Gelscan Results on a Typical Formulated Sample

Sample HPLC Gelscan A 5.0 Χ 10 ug/g 5.4 X 1(P ug/g Β 6.2 X KPug/g o.lXlO^g/g —£— 5,0 X1Q ug/g g^Xl^yg/g The difference between the values is within the expected error of the Gelscan assay. 3

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More work to address, precision and recovery studies, and a morerigorouscorrelation of the HPLC assay to quantitation by SDS-PAGE is underway. Summary In this paper we have described the development of an assay for Bacillus thuringiensis var. san diego toxin using RP-HPLC. The protein is dissolved by raising the pH and then is stabilized by heating in the presence of 50% ethylene glycol. Calibration curves based on peak area of purified toxin are linear and reproducible. The assay requires only a few hours, replacing an SDS-PAGE based assay which ran overnight. Two aspects of the assay are especially interesting. First is the increased recovery from the column by the addition of ethylene glycol to the mobile phase. The mechanism is unclear, but perhaps the glycol masks residual silanol groups or inhibits protein interaction with the bonded



Figure 3:

No Heat

e

40

e

50"

80

e

100°

Gradient: Sample:

Mobile Phase:

A- Water, 2% ethylene glycol, 0.1% TFA Β - Acetonitrile, 2% ethylene glycol, 0.1% TFA 10 minutes at 40% B, gradient to 70% Β in 30 minutes BTSD crystals, dissolved in NaOH, SDS, 50% ethylene glycol, heated 15 mintues

Effect of Heating the Delta-Endotoxin Sample in Ethylene Glycol

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u g T o x i n Injected

Figure 4.

Bacillus thuringiensis var. san diego delta-endotoxin Calibration Curve


8. WITTWERETAL. An HPLC Assayfor a δ-Endotoxin

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I

Ethylene Glycol

SDS/GIycerol

aX + bY-j = c aX2 + bY2 = c 1

Figure 5.

X : First Peak Height Y = Second Peak Height c = ug Toxin Injected

Quantitation of BTSD toxin

phase. Second is the effect of 50% ethylene glycol and heat on the retention time. Based on the effect of added SDS, it appears the glycol stabilizes the toxin in a relatively native state. Literature Cited 1. Edwards, D. L.; Herrnstadt, C; Soares, G. G.; Wilcox, E. R. Bio/Technology 1986, 4, 305-308. 2. Bennet, B. D.; Gaertner, F. H.; Gilroy, T. E.; Herrnstadt, C.; Sobieski, D. A. Gene 1987, 57, 37-46. 3. Andrews, A. T. Electrophoresis, Theory, Techniques and Biochemical and Clinical Applications: 2nd Edition: Clarendon Press Oxford, 1986. 4. Private communication, Sept. 1989, Pat Nolan, Mycogen Corp. RECEIVED March 5, 1990


Analytical Chemistry of Bacillus thuringiensis

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