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Development of patatin knockdown potato tubers using RNA interference (RNAi) technology, for the production of human-therapeutic glycoproteins.

Kim YS, Lee YH, Kim HS, Kim MS, Hahn KW, Ko JH, Joung H, Jeon JH - BMC Biotechnol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Genome Research Center, KRIBB, Daejeon 305-806, Korea. yoon1920@kribb.re.kr

ABSTRACT

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.

Conclusion: 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.

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Analysis of ConA purified glycoproteins in WT and patatin-hpRNAi lines. (A) Determination of total soluble proteins and ConA purified glycoproteins by Bradford assay. One gram of fresh weight of tuber was used for the extraction of total soluble proteins and purification of glycoprotein. (B) SDS-PAGE analysis of ConA purified glycoproteins in WT and patatin-hpRNAi line 4 and 8. Extract (total soluble proteins), ConA-nonbinding and ConA-binding fractions were separated on 10% SDS-PAGE gel and stained with coomassie blue. Patatin was found to be approximately 40 kDa in size in wild-type plants only. Lane M, molecular mass marker. 20 μg of each fraction was loaded in each well. Marker sizes (in kDa) are indicated.
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Figure 5: Analysis of ConA purified glycoproteins in WT and patatin-hpRNAi lines. (A) Determination of total soluble proteins and ConA purified glycoproteins by Bradford assay. One gram of fresh weight of tuber was used for the extraction of total soluble proteins and purification of glycoprotein. (B) SDS-PAGE analysis of ConA purified glycoproteins in WT and patatin-hpRNAi line 4 and 8. Extract (total soluble proteins), ConA-nonbinding and ConA-binding fractions were separated on 10% SDS-PAGE gel and stained with coomassie blue. Patatin was found to be approximately 40 kDa in size in wild-type plants only. Lane M, molecular mass marker. 20 μg of each fraction was loaded in each well. Marker sizes (in kDa) are indicated.

Mentions: Patatin-knockdown potatoes are likely to have a few advantages in terms of glycoprotein purification, compared to wild-type potatoes. To confirm the possible advantages of patatin-knockdown potatoes, potato glycoproteins were purified by affinity chromatography on Sepharose-coupled concanavalin A (ConA) and the purified glycoproteins were monitored by running a SDS-PAGE gel (Figure 5). The content of total soluble proteins was not reduced in transgenic plants, though patatin was almost completely knocked down. In our purification system, the yields of glycoproteins were 32.6% (wild-type), 21.9% (line 4) and 19.3% (line 8) (Figure 5A). The level of glycoproteins in WT was higher than transgenic plants. In addition, SDS-PAGE analysis showed that the fraction of purified glycoproteins in wild-type potatoes has two major bands (Figure 5B). One was patatin and the other was a band in size 26–30 kDa. In contrast, patatin-RNAi lines had only one major band (26–30 kDa). Therefore, no further purification steps to remove patatin were required.


Development of patatin knockdown potato tubers using RNA interference (RNAi) technology, for the production of human-therapeutic glycoproteins.

Kim YS, Lee YH, Kim HS, Kim MS, Hahn KW, Ko JH, Joung H, Jeon JH - BMC Biotechnol. (2008)

Analysis of ConA purified glycoproteins in WT and patatin-hpRNAi lines. (A) Determination of total soluble proteins and ConA purified glycoproteins by Bradford assay. One gram of fresh weight of tuber was used for the extraction of total soluble proteins and purification of glycoprotein. (B) SDS-PAGE analysis of ConA purified glycoproteins in WT and patatin-hpRNAi line 4 and 8. Extract (total soluble proteins), ConA-nonbinding and ConA-binding fractions were separated on 10% SDS-PAGE gel and stained with coomassie blue. Patatin was found to be approximately 40 kDa in size in wild-type plants only. Lane M, molecular mass marker. 20 μg of each fraction was loaded in each well. Marker sizes (in kDa) are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335101&req=5

Figure 5: Analysis of ConA purified glycoproteins in WT and patatin-hpRNAi lines. (A) Determination of total soluble proteins and ConA purified glycoproteins by Bradford assay. One gram of fresh weight of tuber was used for the extraction of total soluble proteins and purification of glycoprotein. (B) SDS-PAGE analysis of ConA purified glycoproteins in WT and patatin-hpRNAi line 4 and 8. Extract (total soluble proteins), ConA-nonbinding and ConA-binding fractions were separated on 10% SDS-PAGE gel and stained with coomassie blue. Patatin was found to be approximately 40 kDa in size in wild-type plants only. Lane M, molecular mass marker. 20 μg of each fraction was loaded in each well. Marker sizes (in kDa) are indicated.
Mentions: Patatin-knockdown potatoes are likely to have a few advantages in terms of glycoprotein purification, compared to wild-type potatoes. To confirm the possible advantages of patatin-knockdown potatoes, potato glycoproteins were purified by affinity chromatography on Sepharose-coupled concanavalin A (ConA) and the purified glycoproteins were monitored by running a SDS-PAGE gel (Figure 5). The content of total soluble proteins was not reduced in transgenic plants, though patatin was almost completely knocked down. In our purification system, the yields of glycoproteins were 32.6% (wild-type), 21.9% (line 4) and 19.3% (line 8) (Figure 5A). The level of glycoproteins in WT was higher than transgenic plants. In addition, SDS-PAGE analysis showed that the fraction of purified glycoproteins in wild-type potatoes has two major bands (Figure 5B). One was patatin and the other was a band in size 26–30 kDa. In contrast, patatin-RNAi lines had only one major band (26–30 kDa). Therefore, no further purification steps to remove patatin were required.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Plant Genome Research Center, KRIBB, Daejeon 305-806, Korea. yoon1920@kribb.re.kr

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus