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In previous studies, we reported that the 3019 protein originating from the silkworm inhibited apoptosis in mammalian cells. In this work, we demonstrated that the 30Kc19 protein, which is most abundant 30K protein in silkworm hemolymph, also enhanced enzyme stability. When the recombinant 30Kc19 protein was supplemented into distilled-deionized water containing alkaline phosphatase or horseradish peroxidase, deactivation of both enzymes induced by non-buffered DDW was significantly suppressed. The increase in enzyme stability due to the presence of 30Kc19 was similar to that observed for bovine serum albumin, which is commonly used in conventional enzyme reactions. The decrease in enzyme activity due to long-term storage in different buffer systems was also inhibited by 30Kc19. The 30Kc19 protein structure was shown to play a vital role in stabilizing the enzyme. These results imply that the recombinant 30Kc19 protein hold promise for use as an additive to increase or maintain enzyme activity. (C) 2011 Elsevier Ltd. All rights reserved. ; This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2010K001137, 2010-0000825, 2010-0020821). This work was also supported by WCU (World Class University) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (R32-2010-000-10213-0). ; OAIID:oai:osos.snu.ac.kr:snu2012-01/102/0000002410/1 ; SEQ:1 ; PERF_CD:SNU2012-01 ; EVAL_ITEM_CD:102 ; USER_ID:0000002410 ; ADJUST_YN:Y ; EMP_ID:A002014 ; DEPT_CD:458 ; CITE_RATE:2.414 ; FILENAME:Stabilization of enzymes by the recombinant 30Kc19 protein.pdf ; DEPT_NM:화학생물공학부 ; EMAIL:thpark@snu.ac.kr ; SCOPUS_YN:Y ; CONFIRM:Y

Cell-penetrating protein and its protein transduction domain have been used to deliver drugs and proteins into the cells via receptor-independent endocytosis. A number of cell-penetrating proteins including TAT derived from HIV-1 virus, VP22 from herpes simplex virus and Antennapedia from drosophila have been discovered. Here, we report a cell-penetrating protein, 30Kc19, originating from the hemolymph of silkworm, Bombyx mori. The 30Kc19 is the first cell-penetrating protein that has been found in insect hemolymph. When the 30Kc19 protein produced from recombinant Escherichia coil was supplemented into the medium for mammalian cell culture, 30Kc19 efficiently penetrated into various types of cells and localized at subcellular compartments including mitochondria and cytoplasm. 30Kc19 also delivered cargo proteins such as green fluorescence protein into the cells even though cargo proteins are not able to penetrate into cells by themselves. In addition to the in vitro system, 30Kc19 exhibited the protein transduction property in vivo. When 30Kc19 was intraperitoneally injected into mice, 30Kc19 delivered cargo proteins into various organ tissues of model animals without producing toxicity. Therefore, 30Kc19 has a great potential as a cell-penetrating protein that can be used as a medicinal tool to deliver cargo molecules including proteins into the target organ tissues in the body. (C) 2012 Elsevier Ltd. All rights reserved. ; This work was supported by the National Research Foundation of Korea (NRF) grant funded by the government of Korea (MEST) (No. 2012K0001365, 2012-0000144, 20110020984, 2010-0002520) and by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea. (Grant No. A103017). This work was also supported by the Pioneer Research Program (No. 2012-0001020) and WCU (World Class University) Program (R32-2011-000-10213-0) through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science, and Technology (MEST). ; OAIID:oai:osos.snu.ac.kr:snu2012-01/102/0000002410/15 ; SEQ:15 ; PERF_CD:SNU2012-01 ; EVAL_ITEM_CD:102 ; USER_ID:0000002410 ; ADJUST_YN:Y ; EMP_ID:A002014 ; DEPT_CD:458 ; CITE_RATE:7.404 ; FILENAME:(2012.12) A protein delivery system using 30Kc19 cell-penetrating protein originating from.pdf ; DEPT_NM:화학생물공학부 ; EMAIL:thpark@snu.ac.kr ; SCOPUS_YN:Y ; CONFIRM:Y

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins. ; This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (nos. 2011-0000331, 2011K000682, and 2011-0020984). This work was also supported by the Pioneer Research Program and by Basic Science Research Program through the NRF grant funded by the Ministry of Education, Science, and Technology (nos. 2011-0001643 and 2010-0002520). This work was also supported by WCU (World Class University) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (R32-2010-000-10213-0) and by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (grant no. A103017). ; OAIID:oai:osos.snu.ac.kr:snu2012-01/102/0000002410/14 ; SEQ:14 ; PERF_CD:SNU2012-01 ; EVAL_ITEM_CD:102 ; USER_ID:0000002410 ; ADJUST_YN:Y ; EMP_ID:A002014 ; DEPT_CD:458 ; CITE_RATE:3.425 ; FILENAME:(2012.11)Enhancement of recombinant human EPO production and.pdf ; DEPT_NM:화학생물공학부 ; EMAIL:thpark@snu.ac.kr ; SCOPUS_YN:Y ; CONFIRM:Y