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High-level production of bioactive heterodimeric protein human interleukin-12 in rice

Paper ID Volume ID Publish Year Pages File Format Full-Text
17699 42690 2010 5 PDF Available
Title
High-level production of bioactive heterodimeric protein human interleukin-12 in rice
Abstract

Human interleukin-12 (hIL-12), a heterodimeric cytokine comprised of p35 and p40 subunits, was generated previously via tobacco cell suspension culture at a maximum concentration of only 175 μg/L. In order to improve the productivity, the rice α-amylase 3D promoter (Ramy3D) was utilized in order to express recombinant protein at high levels under sucrose starvation conditions. The hIL-12 gene was cloned into the rice expression vector under the control of the Ramy3D promoter. Transgenic rice calli were prepared via particle bombardment-mediated transformation and were screened for rhIL-12p70 expression using ELISA. Western blot and bioassay results confirmed that rhIL-12p70 was expressed and secreted into the culture medium as a functionally active heterodimeric form, and the productivity of rhIL-12p70 was estimated as 12.7 mg/L, which is approximately 73-fold higher than was observed with the tobacco cell culture system [Kwon TH, Seo JE, Kim J, Lee JH, Jang YS, Yang MS. Expression and secretion of the heterodimeric protein interleukin-12 in plant cell suspension culture. Biotechnol Bioeng 2003;81:870–5]. However, the secreted rhIL-12p70 proved unstable in the medium, and degraded rapidly after day 13. In order to stabilize the secreted rhIL-12p70, three stabilizing polymers were tested (polyethylene glycol, polyvinylpyrrolidone, and gelatin). Gelatin was the most effective in stabilizing the secreted rhIL-12p70. After the addition of 2% (w/v) gelatin, the maximum rhIL-12p70 concentration reached 31.0 mg/L, representing a 2.5-fold increase over the control.

Keywords
Interleukin-12; Rice cell suspension culture; Rice α-amylase promoter; Sucrose starvation
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High-level production of bioactive heterodimeric protein human interleukin-12 in rice
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 46, Issue 5, 5 April 2010, Pages 347–351
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us