Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor.
In this study, we synthesized a synthetic serine proteinase inhibitor II gene (sPI-II) that harbored the chymotrypsin and trypsin inhibitor domains of the PI-II gene from Nicotiana alata. In an effort to reduce protease activity in a rice cell suspension culture, we first synthesized sPI-II using overlap PCR and then introduced the gene into a rice calli (Oryza sativa L. cv. Dongin) by particle bombardment-mediated transformation. The sPI-II gene was under the control of a rice alpha-amylase 3D promoter induced by sugar starvation. To verify the integration and expression of the sPI-II gene in the transformed rice cells, we employed genomic DNA PCR amplification and Northern blot analysis, respectively. The relative protease activity of the transformed cell suspension culture was reduced to approximately 23% when compared to the non-transformed culture. This indicates that a transformed suspension culture system expressing a proteinase inhibitor, may be a useful tool to protect against recombinant protein losses resulting from extracellular proteases.
Kim
Tae-Geum
TG
Division of Biological Sciences and the Research Center for Bioactive Materials, Chonbuk National University, Jeonju 561-756, Republic of Korea.
Kim
Hyang-Mi
HM
Lee
Ho-Jin
HJ
Shin
Yun-Ji
YJ
Kwon
Tae-Ho
TH
Lee
Nan-Ju
NJ
Jang
Yong-Suk
YS
Yang
Moon-Sik
MS
D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D005822:Genetic Vectors; D012275:Oryza / Q000235:genetics* / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D017931:DNA Primers; D018744:DNA, Plant; C075489:PI-I protein, Nicotiana tabacum; D010940:Plant Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases