APOPLAST WASH FLUID RECOVERY FOR IMPROVED RECOMBINANT ENDOGLUCANASE PRODUCTION IN TOBACCO LEAVES
First Claim
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1. A method of producing E1 endoglucanase in a leaf tissue, the method comprising:
- providing leaf tissue from Nicotiana tabacum transiently transformed to express E1 endoglucanase from Acidothermus cellulolyticus under the control of the CaMV 35S promoter;
incubating the leaf tissue at 20°
C. in a humid environment such that the E1 endoglucanase is expressed and located to an apoplast of a cell of the leaf tissue;
contacting the leaf tissue with a rinse fluid using vacuum infiltration-centrifugation once every 24 hours over the course of 6 days to release the E1 endoglucanase from the apoplast into the rinse fluid to create an apoplast wash fluid;
wherein content of the E1 endoglucanase in the apoplast wash fluid from leaf tissue contacted with rinse fluid once every 24 hours for 6 days is at least two-fold higher than the content of the E1 endoglucanase in the apoplast wash fluid from a comparable leaf tissue contacted with rinse fluid only at the end of the 6 days.
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Abstract
The present disclosure relates to methods of increasing the production of an apoplast-targeted recombinant protein in a plant tissue and to methods of increasing the purity of an apoplast-targeted recombinant protein recovered from a plant tissue.
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Citations
1 Claim
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1. A method of producing E1 endoglucanase in a leaf tissue, the method comprising:
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providing leaf tissue from Nicotiana tabacum transiently transformed to express E1 endoglucanase from Acidothermus cellulolyticus under the control of the CaMV 35S promoter; incubating the leaf tissue at 20°
C. in a humid environment such that the E1 endoglucanase is expressed and located to an apoplast of a cell of the leaf tissue;contacting the leaf tissue with a rinse fluid using vacuum infiltration-centrifugation once every 24 hours over the course of 6 days to release the E1 endoglucanase from the apoplast into the rinse fluid to create an apoplast wash fluid; wherein content of the E1 endoglucanase in the apoplast wash fluid from leaf tissue contacted with rinse fluid once every 24 hours for 6 days is at least two-fold higher than the content of the E1 endoglucanase in the apoplast wash fluid from a comparable leaf tissue contacted with rinse fluid only at the end of the 6 days.
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Specification