1. A method for recovering apoplast-targeted recombinant protein from plant tissue, the method comprising:
a first step of providing plant tissue transformed to express an apoplast-targeted recombinant protein;
a second step of contacting the plant tissue with a cell wall-degrading enzyme;
a third step of extracting the apoplast-targeted recombinant protein from the plant tissue;
a fourth step of incubating the plant tissue for an incubation period; and
a fifth step of extracting the apoplast-targeted recombinant protein from the plant tissue,
wherein the fifth step extracts the apoplast-targeted recombinant protein synthesized after the third step.
2. The method of claim 1, wherein the third step of extracting the apoplast-targeted recombinant protein from the plant tissue comprises contacting the plant tissue with a rinse fluid to release the apoplast-targeted recombinant protein from the apoplast into the rinse fluid to create an apoplast wash fluid.
3. The method of claim 1, wherein the third step of extracting the apoplast-targeted recombinant protein from the plant tissue comprises contacting the plant tissue with a rinse fluid on a plurality of occasions over the course of a production interval to release the apoplast-targeted recombinant protein from the apoplast into the rinse fluid to create an apoplast wash fluid, where the content of the apoplast-targeted recombinant protein in the apoplast wash fluid from the plant tissue contacted with the rinse fluid on the plurality of occasions over the course of the production interval is higher than the content of the apoplast-targeted recombinant protein in the apoplast wash fluid from comparable plant tissue contacted with the rinse fluid only at the end of the production interval.
4. The method of claim 1, wherein the method further comprises a sixth step of contacting the plant tissue with the cell wall-degrading enzyme.
5. The method of claim 1, wherein the cell wall-degrading enzyme is pectinase.
6. The method of claim 5, wherein the pectinase is at a concentration of about 0.15% vv.
7. The method of claim 5, wherein the pectinase is at a concentration ranging from about 0.10% to about 0.2% or from about 0.05% to about 0.25%.
8. The method of claim 1, wherein the cell wall-degrading enzyme is selected from the group consisting of cellulases, hemicellulases, pectinases, endoglucanases, and exoglucanases.
9. The method of claim 1, wherein the incubation period is selected from the group consisting of 12 hours, 1 day, and 2 days.
10. The method of claim 1, wherein the plant tissue is leaf tissue.
11. The method of claim 1, wherein the plant tissue is transiently transformed using Agrobacterium.
12. The method of claim 1, wherein the apoplast-targeted recombinant protein is at least about 51 kDa to about 340 kDa.
13. The method of claim 1, wherein the apoplast-targeted recombinant protein forms an oligomer.
14. The method of claim 1, wherein the second step of contacting the plant tissue with a cell wall-degrading enzyme takes place at the same time as the third step of extracting the apoplast-targeted recombinant protein from the plant tissue.
15. The method of claim 1, wherein the amount of the apoplast-targeted recombinant protein recovered from the plant tissue is at least six-fold higher than the amount of the apoplast-targeted recombinant protein recovered from the plant tissue not treated with the cell wall-degrading enzyme.
16. The method of claim 1, wherein the purity of the apoplast-targeted recombinant protein recovered from the plant tissue is at least two-fold higher than the amount of the apoplast-targeted recombinant protein recovered from the plant tissue not treated with the cell wall-degrading enzyme.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A password providing system configured to issue a password for use in accessing age-restricted content, comprising at least one processor configured to be connected to a network and configured to:
receive a request for a password from a user computer on a network;
receive from the user computer, over the network, a still or video image of a user of the user computer, the image of the user provided by a camera associated with the user computer;
receive an image of an identification document having identification information associated with the user of the user computer, wherein the image of the identification document is distinct from the still or video image of the user;
cause display of the still or video image and the image of the identification document on a display device associated with the at least one processor to allow an operator to compare the still or video image against the identification information on the identification document and make a determination from that comparison if the user of the user computer is of an appropriate age to access age-restricted content accessible using the password; and
transmit the password to the user computer upon a determination by the operator that the user of the user computer is of an appropriate age to access the age-restricted content accessible using the password.
2. A system as claimed in claim 1, wherein the at least one processor is further configured to associate a time period with the password, the password being valid during the time period and expiring upon expiration of the time period.
3. A system as claimed in claim 1, wherein the identification document includes a picture of the user of the user computer and wherein the at least one processor is further configured to cause display of, on the display device associated with the at least one processor, an image of the picture of the user from the identification document to allow the operator to compare the still or video image against the picture from the identification document and make a determination from that comparison if the age of the user of the user computer is likely appropriate for receipt of the password.
4. A system as claimed in claim 1, wherein the identification information includes age information associated with the age of the user of the user computer and wherein the at least one processor is further configured to cause display of, on the display device associated with the at least one processor, an image of the age information from the identification document to allow the operator to compare the still or video image against the age information from the identification document and make a determination from that comparison if the age of the user of the user computer is likely appropriate for receipt of the password.
5. A system as claimed in claim 1, wherein the identification document comprises a driver’s license.
6. A system as claimed in claim 1, wherein the at least one processor is further configured to allow access to age-restricted content to a user having a valid password and inhibit access to age-restricted content to a user having an expired password.
7. A system as claimed in claim 1, wherein the at least one processor is configured to receive the image of the identification document from the user computer over the network.
8. A system as claimed in claim 1, wherein the at least one processor employs a videoconferencing communication connection with the user computer to receive the still or video image of the user.
9. A system as claimed in claim 1, wherein the at least one processor employs a videoconferencing communication connection with the user computer to receive the still or video image of the user and the image of the identification document.
10. A password providing method to issue a password for use in accessing age-restricted content via a network, the method comprising:
receiving, by at least one processor, over a network, a request for a password from a user computer on the network;
receiving, by the at least one processor, from the user computer, over the network, a still or video image of a user of the user computer, provided by a camera associated with the user computer;
receiving an image of an identification document having identification information associated with the user of the user computer, wherein the image of the identification document is distinct from the still or video image of the user;
causing display of the still or video image and the image of the identification document on a display device associated with the at least one processor to allow an operator to compare the still or video image against the identification information on the identification document and make a determination from that comparison if the user of the user computer is of an appropriate age to access the age-restricted content accessible using the password; and
transmitting the password to the user computer upon a determination by the operator that the user of the user computer is an appropriate age to access age-restricted content accessible using the password.
11. A method as claimed in claim 10, further comprising associating a time period with the password, the password being valid during the time period and expiring upon expiration of the time period.
12. A method as claimed in claim 10, wherein the identification document includes a picture of the user of the user computer and wherein causing display of the image of the identification document on the display device associated with the at least one processor comprises causing display of an image of the picture of the user from the identification document to allow the operator to compare the still or video image against the picture from the identification document and make a determination from that comparison if the user of the user computer is likely of an appropriate age to access the age-restricted content accessible using the password.
13. A method as claimed in claim 10, wherein the identification information includes age information associated with the age of the user of the user computer and wherein causing display of the image of the identification document on the display device associated with the at least one processor comprises causing display of an image of the age information from the identification document to allow the operator to compare the still or video image against the age information from the identification document and make a determination from that comparison if the user of the user computer is likely of an appropriate age to access the age-restricted content accessible using the password.
14. A method as claimed in claim 10, wherein the identification document comprises a driver’s license.
15. A method as claimed in claim 10, further comprising allowing access to age-restricted content to a user having a valid password and inhibiting access to age-restricted content to a user having an expired password.
16. A method as claimed in claim 10, wherein receiving the image of the identification document comprises receiving the image of the identification document from the user computer over the network.
17. A method as claimed in claim 10, wherein the at least one processor employs a videoconferencing communication connection with the user computer to receive the still or video image of the user.
18. A method as claimed in claim 10, wherein the at least one processor employs a videoconferencing communication connection with the user computer to receive the still or video image of the user and the image of the identification document.
19. A method as claimed in claim 10, wherein the user computer is a mobile, hand-held device.
20. A non-transitory computer readable storage medium having stored thereon, instructions that, if executed by a computing device, cause the computing device to perform operations of issuing a password for use in accessing age-restricted content, the operations comprising:
receiving a request for a password from a user computer on a network;
receiving from the user computer, over the network, a still or video image of a user of the user computer, provided by a camera associated with the user computer;
receiving an image of an identification document having identification information associated with the user of the user computer, wherein the image of the identification document is distinct from the still or video image of the user;
causing display of the still or video image and the image of the identification document on a display device to allow an operator to compare the still or video image against the identification information on the identification document and make a determination from that comparison if the user of the user computer is of an appropriate age to access age-restricted content accessible using the password; and
transmitting the password to the user computer upon a determination by the operator that the user of the user computer is an appropriate age to access the age-restricted content accessible using the password.
21. The computer readable storage medium of claim 20, the operations further comprising associating a time period with the password, the password being valid during the time period and expiring upon expiration of the time period.
22. The computer readable storage medium of claim 20, wherein the identification document includes a picture of the user of the user computer and wherein causing display of the image of the identification document on the display device comprises causing display of an image of the picture of the user from the identification document to allow the operator to compare the still or video image against the picture from the identification document and make a determination from that comparison if the user of the user computer is likely of an appropriate age to access the age-restricted content accessible using the password.
23. The computer readable storage medium of claim 20, wherein the identification information includes age information associated with the age of the user of the user computer and wherein causing display of the image of the identification document on the display device comprises causing display of an image of the age information from the identification document to allow the operator to compare the still or video image against the age information from the identification document and make a determination from that comparison if the user of the user computer is likely of an appropriate age to access the age-restricted content accessible using the password.
24. The computer readable storage medium of claim 20, wherein the identification document comprises a driver’s license.
25. The computer readable storage medium of claim 20, the operations further comprising allowing access to age-restricted content to a user having a valid password and inhibiting access to age-restricted content to a user having an expired password.
26. The computer readable storage medium of claim 20, wherein receiving the image of the identification document comprises receiving the image of the identification document from the user computer over the network.
27. The computer readable storage medium of claim 20, wherein the user computer is a mobile, hand-held device.