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High‐level transient expression of recombinant protein in lettuce
Author(s) -
Joh Lawrence D.,
Wroblewski Tadeusz,
Ewing Nicholas N.,
VanderGheynst Jean S.
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20557
Subject(s) - agrobacterium tumefaciens , incubation , agroinfiltration , horticulture , darkness , incubation period , biology , infiltration (hvac) , recombinant dna , gene expression , chemistry , microbiology and biotechnology , botany , biochemistry , gene , transgene , materials science , composite material
Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the β‐glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10 9 colony forming units/mL followed by incubation for 72 h at 22°C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations ≤100 ppm in the vacuum infiltration did not affect GUS expression, while infiltration with an A. tumefaciens density of 10 7 and 10 8 colony forming units/mL, incubation at 29°C, and a surfactant concentration of 1,000 ppm significantly reduced expression. Incubation in continuous light caused lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22°C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi‐quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R 2 value of 0.90 was determined between log 10 (% leaf darkness) versus log 10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high‐value recombinant proteins in planta. © 2005 Wiley Periodicals, Inc.