APOPLAST WASH FLUID RECOVERY FOR IMPROVED RECOMBINANT ENDOGLUCANASE PRODUCTION IN TOBACCO LEAVES

• US 20140106434A1
• Filed: 10/11/2013
• Published: 04/17/2014
• Est. Priority Date: 10/11/2012
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: MCDONALD, Karen A., KINGSBURY, Nathaniel J.
• Granted Patent: US 9,617,297 B2
• Time in Patent Office: Days
• US Class Current: 435/209
• CPC Class Codes: C07K 1/14 Extraction; Separation; Purification
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

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.

Claims

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.