Protein expression using an Agrobacterium-mediated Transient Plant Production system
All expression constructs were mobilized into Agrobacterium tumefaciens strain LBA4404 using a freeze/thaw method (Holsters et al., 1978). Transformed A. tumefaciens (Agro ) was grown in 5 ml of YEP medium [10 g/L Bacto-peptone (Difco), 10 g/L yeast extract (Difco), 5 g/L NaCl (Sigma-Aldrich), pH 7.0] containing 0.1 g/L of kanamycin (Sigma-Aldrich) for antibiotic selection of the expression construct and 0.06 g/L of streptomycin (Sigma-Aldrich) for selection of the T-DNA plasmid. A. tumefaciens cultures were grown at 28°C on an orbital shaker at 225 rpm for 2 days. The 5 ml bacterial suspensions were used to inoculate 50 ml of YEP medium containing the antibiotics and cultured for an additional 24 h. These A. tumefacienscultures were diluted into induction medium (IM; 10mM MES pH 5.5) at an OD600 of 0.2.
Transient expression for all constructs in Nicotianabenthamiana plants was carried out using anAgrobacterium -mediated vacuum infiltration method (Agro -infiltration) previously described by Medrano et al (2009). Briefly, 4-6-week-old N. benthamiana plants were grown and maintained under controlled temperature, light and humidity. Plants were vacuum infiltrated with A . tumefaciens cultures containing the cfIL-22 gene or pBK (empty vector; plant background control). TheA. tumefaciens -infiltrated tobacco plants were returned to the environmental growth chamber and leaf tissues were collected at 2-7 days post infiltration to determine harvest time providing maximum protein accumulation. All leaf tissue was frozen in liquid nitrogen and stored at -80°C until further analysis.