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Data Evaluation Report - In Vitro Digestibility MRID NO: 44258108
Date: 1/6/98
Reviewed by: John L. Kough,
Ph.D., Biologist, BPPD
Secondary Reviewer: Doug Gurian-Sherman, Ph.D., Plant Pathologist,
BPPD
STUDY TYPE: In Vitro Digestibility
MRID NO: 44258108
CHEMICAL NO: 006466
TEST MATERIAL: Cry9C delta-endotoxin of Bacillus thuringiensis
STUDY NO: none assigned
SPONSOR: Plant Genetic Systems (America) Inc., Des Moines, IA
TEST FACILITY: Plant Genetic Systems N.V., Ghent, Belgium
TITLE OF REPORT: In Vitro Digestibility and Heat Stability of the Endotoxin Cry9C of Bacillus thuringiensis
AUTHOR: Marnix Peferoen
STUDY COMPLETED: March 28, 1997
CONCLUSION: The samples of lyophilized Cry9C protein expressed in corn showed no signs of protein disintegration when subjected to in vitro digestion in simulated mammalian gastric fluid. These digestions were done either with or without pepsin in the low pH buffer and were assayed by western blot from samples taken at several time points from the mixing the reagents to after 4 hours exposure to the digestive fluids. The same 70kD double band seen in the original Cry9C protein in plant tissue at time 0 was also seen, undiminished, in all the subsequent incubation samples. No effect on Cry9C activity as determined by bioassay was seen after any heat treatment. The most stringent heat treatment was 90ºC for 10 minutes.
CLASSIFICATION: ACCEPTABLE.
STUDY DESIGN
Purified Cry9C delta-endotoxin as expressed in corn was examined for stability to heat and in vitro digestion in simulated gastric fluid. This study has a GLP statement indicating non-compliance but no reasons were given.
TEST METHODS
Test Substance
Cry9C protein purified from Bacillus thuringiensis and as found in lyophilized corn tissue was used as the test substance in these studies. The CryIA(b) endotoxin purified from fermentation of an E. coli strain containing the CryIA(b) gene was also used as a reference substance for other delta-endotoxins. The methods to prepare these substances is discussed below.
PREPARATION OF CRY9C PROTEIN IN Bacillus thuringiensis
A B. thuringiensis strain cured of its indigenous plasmids and transformed with plasmid pGI9CK was grown in fermentation to yield a culture that was pelleted by centrifugation. Half the pellets were lyophilized and stored at -20EC, the other half stored at 4EC. When needed, the bacterial powder was solubilized in alkaline buffer (0.04M Na2CO3-HCl; 0.01M DDT; 5mM EDTA; 0.1mM PMSF; pH 10) preserved with 0.02% NaN3 and stirred for 4 hours at 4EC. The solution is clarified by centrifugation (20 minutes at 12,000 rpm in a Sorvall SA-600) then dialyzed in multiple fraction against a buffer (20mM TRIS/HCl; 25mM NaCl; 5mM EDTA, pH8.6). The dialysis tubing was a spectra-por #4 with a cutoff of 12-14kDa. The multiple fraction samples were then diluted with the dialysis buffer to 0.5 mg protein/ml and treated with trypsin (0.05%w/v) for 1 hour at 37EC. The trypsin digest was stopped by the addition of PMSF to yield a 1mM final concentration. All the samples, still separated, were then pooled, mixed and subjected to an ammonium sulfate precipitation (final concentration of 3M or 77% saturation) overnight at 4EC. The protein was pelleted out by centrifugation (20 minutes at 12,000 rpm in a Sorvall SA-600), dissolved in 20mM ethanolamine/HCl (pH9.5) and a protein determination done. The solution was again dialyzed in multiple fractions against 20mM ethanolamine with 2 changes, then against 10mM ethanolamine (pH9.5) with 2 changes. Finally the samples are lyophilized and the protein concentration of the powder determined.
PREPARATION OF CRY9C POWDER FROM CORN PLANTS
Seeds, marked CBH-351 from a segregating population of transformed plants, were grown for six weeks, leaf samples removed and bioassayed for activity against European corn borer. Four weeks later, the same plants were assayed for Cry9c by an ELISA technique (SOP PGS-9D/1, not provided). In the presence of conflicting results between the two assays, that particular corn plant was not used for further processing. Ten week old plants were harvested, the leaves and stalks were cut into 10cm sections for lyophilization. Roots were also harvested, washed and cut into 2-5cm fragments. The individual plant samples were lyophilized, blended to reduce particle size, transferred to plastic bottles and stored in the dark at room temperature. Prior to use a 50mg sample of the plant powder was removed and the proteins extracted (SOP PGS-9H/0, not provided). The protein concentration was determined (Bradford Microassay SOP PGS-9E/0, not provided) and presence of Cry9C determined by ELISA (SOP PGS9D/1, not provided) and bioassay against European corn borer on artificial diet (SOP PGS-9A/1, not provided).
PREPARATION OF CRY1Ab PROTEIN FROM Escherichia coli
Cry1Ab protein was recovered from a recombinant strain of E. coli containing the Cry1Ab gene. The purification process for the protoxin and activation to toxin are described in a cited article (Lambert, B.; Buysse, L.; Decock, C.; Jansens, S.; Piens, C.; Saey, B.; Seurinck, J.; van Audenhove, K.; van Rie, J.; van Vliet, A. & Peferoen, M. (1996) A Bacillus thuringiensis insecticidal crystal protein with a high activity against members of the family Noctuidae. Applied and Environmental Microbiology 62, 80-86).
Test System
In vitro DIGESTION
Samples of tissue containing Cry9C protein or purified Cry9C protein were dissolved in simulated gastric fluid with pepsin (0.32% pepsin in gastric buffer (0.5gm NaCl, 1.75ml of 1M HCl in 250ml water, pH2.0)) and incubated at 37ºC. Samples were removed for western blot analysis at 0, 5, 15, 60 and 240 minutes of incubation. No pepsin inhibitor was added prior to preparation for SDS-PAGE.
HEAT STABILITY
Samples containing Cry9C or Cry1Ab were dissolved in TRIS buffer (20mM TRIS/HCl, 25mM NaCl, 5mM EDTA, pH8.6) and heated for 10 minutes in a water bath at 70, 80 or 90ºC. The samples were then tested for insecticidal activity in a European corn borer bioassay (SOP PGS-9A/1, not provided). Two studies were done to examine the effect of heat treatment. The first was done with 500μg/ml of Cry9C and the bioassay was done at a single dose of 1250ng/cm2 diet surface. The second assay was done with both Cry9C and Cry1Ab at 50μg/ml and tested at either 100ng/cm2 or 1250ng/cm2 diet surface.
INSECT BIOASSAYS
Bioassays of endotoxin activity after the various treatments described above were done on individual European corn borer larva in 24 well reading plates supplied with artificial diet (SOP PGS-9A/1, not provided). Details of the assay were not provided here.
RESULTS AND DISCUSSION
The samples of lyophilized Cry9C protein expressed in corn showed no signs of protein disintegration when subjected to in vitro digestion in simulated gastric fluid. Reproductions of the gels are seen in an appendix to this review. These digestions were done either with or without pepsin in the low pH buffer and were assayed by western blot from samples taken at several time points from the mixing the reagents to after 4 hours exposure to the digestive fluids.
The same 70kD double band seen in the original Cry9C protein in plant tissue at time 0 was also seen, undiminished, in all the subsequent incubation samples. Interestingly, the three bands from microbially produced Cry9C (either E. coli or B. thuringiensis) were different from the plant source Cry9C. One band in both microbial preps comigrated with the 70 kD doublet. The addition of pepsin resulted in the appearance of some lower molecular weight bands but the 70 kD double band did not decrease in intensity as in the normal digestion pattern. Typically, evidence of digestion in a western blot is the gradual reduction or disappearance of the high molecular weight bands with increasing amounts of lower weight bands appearing and/or disappearing over time.
The effects of heat treatments on Cry9C protein were tracked by the changes seen in European corn borer bioassays. Two studies were done to examine the effect of heat treatment. The first was done with 500μg/ml of Cry9C and the bioassay was done at a single dose of 1250ng/cm2 diet surface. The second assay was done with both Cry9C and Cry1Ab at 50μg/ml and tested at either 100ng/cm2 or 1250ng/cm2 diet surface. The results of both assays are found in the attached appendix. Basically there was no effect on Cry9C activity by any heat treatment. The second series of assays showed a decline of % mortality of 88 to 79 % with the temperature increase from 70ºto 80º or 90ºC. This slight increase represents two more individual larva succumbing and is probably not a significant finding. The Cry1Ab showed a decrease in activity with increased heat from 42% mortality to essentially background at 80ºC. The stated LC50 values are 37.9 and 128.7 for Cry9C and Cry1Ab, respectively were apparently not done with these samples. No units are stated for these values but they may be the same ng/cm2 as the test system.
The results of these studies indicate the trypsin activated fragment of Cry9C is both stable to gastric digestion and heat to 90ºC. These are two characteristics that are frequently found in proteins that are known to be the cause of food allergies. It is important to note that there are probably numerous proteins in food plants that are both heat stable and resistant to gastric digestion. These two biochemical characteristics are not by themselves the sole indicators that they are likely to be food allergens.