Development of patatin knockdown potato tubers using RNA interference (RNAi) technology, for the production of human-therapeutic glycoproteins.
BACKGROUND
Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.


RESULTS
Based upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence (635 nts) of patatin gene pat3-k1 [GenBank accession no. DQ114421] in potato plants (Solanum tuberosum L.) was amplified for the construction of a patatin-specific hairpin RNAi (hpRNAi) vector. The CaMV 35S promoter-driven patatin hpRNAi vector was transformed into the potato cultivar Desiree by Agrobacterium-mediated transformation. Ten transgenic potato lines bearing patatin hpRNA were generated. The effects of RNA interference were characterized at both the protein and mRNA levels using 1D and 2D SDS/PAGE and quantitative real-time RT-PCR analysis. Dependent upon the patatin hpRNAi line, patatins decreased by approximately 99% at both the protein and mRNA levels. However, the phenotype (e.g. the number and size of potato tuber, average tuber weight, growth pattern, etc.) of hpRNAi lines was not distinguishable from wild-type potato plants under both in vitro and ex vitro growth conditions. During glycoprotein purification, patatin-knockdown potato tubers allowed rapid purification of other potato glycoproteins with less contamination of patatins.


CONCLUSIONS
Patatin-specific hpRNAi effectively suppressed the expression of a majority of patatin variants in potato tubers via the specific degradation of individual mRNAs of the patatin multi-gene family. More importantly, patatin-knockdown potato tubers appear to be an ideal host for the production of human therapeutic glycoproteins, because they eventually allow fast, easy purification of recombinant proteins, with less contamination from potato glycoprotein patatins.
Kim
Yoon-Sik
YS
Plant Genome Research Center, KRIBB, Daejeon 305-806, Korea. yoon1920@kribb.re.kr
Lee
Yong-Hwa
YH
Kim
Hyun-Soon
HS
Kim
Mi-Sun
MS
Hahn
Kyu-Woong
KW
Ko
Jeong-Heon
JH
Joung
Hyouk
H
Jeon
Jae-Heung
JH
D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D024861:Genetic Enhancement / Q000379:methods; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use*; D006801:Humans; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D034622:RNA Interference; D011198:Solanum tuberosum / Q000502:physiology*
D006023:Glycoproteins; D010940:Plant Proteins; C043222:patatin protein, Solanum tuberosum; D002265:Carboxylic Ester Hydrolases