https://repository.kopri.re.kr/handle/201206/5153 2026-04-21T11:40:59Z 2026-04-21T11:40:59Z Lee, Jun Hyuck Kim, Hak Jun Han, Se Jong Yong-Hoe Choe Eunjung Kim Jong Chan Park Do, Hackwon Lee, Sung Gu https://repository.kopri.re.kr/handle/201206/6137 2022-03-24T07:11:31Z 2013-01-01T00:00:00Z

Title: Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris Authors: Lee, Jun Hyuck; Kim, Hak Jun; Han, Se Jong; Yong-Hoe Choe; Eunjung Kim; Jong Chan Park; Do, Hackwon; Lee, Sung Gu Abstract: Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. Because this property of IBPs can increase the freeze-thaw survival of cells, IBPs has attracted the attention from industries for their potential use in biotechnoogical applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris sytem. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25°C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ~100 mg/L. The total amount of secreted proteins at these conditions (25°C, pH 6.0, and glycerol fed-batch phase) was ~443 mg/L, 60 % of which was rLeIBP, yielding ~272 mg/L. In the pilot scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/L. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for six days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42°C, which is almost the same to those reported previously. The avaotechnoogical applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris sytem. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25°C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ~100 mg/L. The total amount of secreted proteins at these conditions (25°C, pH 6.0, and glycerol fed-batch phase) was ~443 mg/L, 60 % of which was rLeIBP, yielding ~272 mg/L. In the pilot scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/L. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for six days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42°C, which is almost the same to those reported previously. The ava

2013-01-01T00:00:00Z Kim, Hak Jun James A. Raymond https://repository.kopri.re.kr/handle/201206/6460 2022-03-24T07:13:55Z 2012-01-01T00:00:00Z

Title: Possible role of horizontal gene transfer in the colonization of sea ice by algae Authors: Kim, Hak Jun; James A. Raymond Abstract: Diatoms and other algae not only survive, but thrive in sea ice. Among sea ice diatoms, all species examined so far produce ice-binding proteins (IBPs), whereas no such proteins are found in non-ice-associated diatoms, which strongly suggests that IBPs are essential for survival in ice. The restricted occurrence also raises the question of how the IBP genes were acquired. Similar proteins are produced by ice-associated bacteria, and so it has previously been speculated that the genes were acquired by horizontal transfer (HGT) from bacteria. Here we report several new IBP sequences from three types of ice algae, which together with previously determined sequences reveal a phylogeny that is completely incongruent with algal phylogeny, and that can be most easily explained by HGT. HGT is also supported by the finding that the closest matches to the algal IBP genes are all bacterial genes and that the algal IBP genes lack introns. We also describe a highly freeze-tolerant bacterium from the bottom layer of Antarctic sea ice that produces an IBP with 44% amino acid identity to a diatom IBP from the same layer, demonstrating at least an opportunity for gene transfer. Together, these results suggest that the success of diatoms and other algae in sea ice can be at least partly attributed to their acquisition of prokaryotic IBP genes.ests that IBPs are essential for survival in ice. The restricted occurrence also raises the question of how the IBP genes were acquired. Similar proteins are produced by ice-associated bacteria, and so it has previously been speculated that the genes were acquired by horizontal transfer (HGT) from bacteria. Here we report several new IBP sequences from three types of ice algae, which together with previously determined sequences reveal a phylogeny that is completely incongruent with algal phylogeny, and that can be most easily explained by HGT. HGT is also supported by the finding that the closest matches to the algal IBP

2012-01-01T00:00:00Z Lee, Sung Gu Kim, Hak Jun Lee, Jun Hyuck Kang, Sung-Ho Koh, Hye Yeon https://repository.kopri.re.kr/handle/201206/6177 2022-03-24T07:11:29Z 2012-01-01T00:00:00Z

Title: Cryopreservative effects of the recombinant ice-binding protein from the Arctic yeast Leucosporidium sp. on red blood cells Authors: Lee, Sung Gu; Kim, Hak Jun; Lee, Jun Hyuck; Kang, Sung-Ho; Koh, Hye Yeon Abstract: Antifreeze proteins (AFPs) have important functions in many freeze-tolerant organisms. The proteins non-colligatively lower the freezing point, and functionally inhibit ice recrystallization in frozen solutions. In our previous studies, we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40% glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze-thaw-induced hemolysis at either rapid- (45°C) or slow-warming (22°C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the ability of LeIBP to inhibit ice recrystallization helps the RBCs avoid critically damaging electrolyte concentrations, which are known as solution effects. Considering all these data, LeIBP can be thought of as a key component in improving RBC cryopreservation efficiency. we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40% glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze-thaw-induced hemolysis at either rapid- (45°C) or slow-warming (22°C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the abil

2012-01-01T00:00:00Z Kim, Hak Jun Sang Hyun Moh Lee, Jun Hyuck Young Min Chi Park, Kyoung Sun Do, Hackwon Ae Kyung Park https://repository.kopri.re.kr/handle/201206/6364 2022-03-24T07:11:23Z 2012-01-01T00:00:00Z

Title: Structural Basis for Antifreeze Activity of Ice-binding Protein from Arctic Yeast Authors: Kim, Hak Jun; Sang Hyun Moh; Lee, Jun Hyuck; Young Min Chi; Park, Kyoung Sun; Do, Hackwon; Ae Kyung Park Abstract: Arctic yeast Leucosporidium sp. produces a glycosylated ice-binding protein (LeIBP) with a molecular mass of approximately 25 kDa, which can lower the freezing point below the melting point once it binds to ice. LeIBP is a member of a large class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57 ？and 2.43 ？resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96？115 form a long α-helix that packs along one face of the β-helix) and a C-terminal hydrophobic loop region (243-PFVPAPEVV-251). Unexpectedly, the C- terminal hydrophobic loop region has an extended conformation pointing away from the body of the coiled structural domain and forms intertwined dimer interactions. In addition, structural analysis of glycosylated LeIBP with sugar moieties attached to Asn185 provides a basis for interpreting previous biochemical analyses as well as the increased stability and secretion of glycosylated LeIBP. We also determined that the aligned Thr/Ser/Ala residues are critical for ice binding within the B face of LeIBP using site-directed mutagenesis. Although LeIBP has a common β-helical fold similar to that of canonical harge class of ice-binding proteins, the structures of which are unknown. Here, we report the crystal structures of non-glycosylated LeIBP and glycosylated LeIBP at 1.57 ？and 2.43 ？resolution, respectively. Structural analysis of the LeIBPs revealed a dimeric right-handed β-helix fold, which is composed of three parts: a large coiled structural domain, a long helix region (residues 96？115 form a long α-helix that packs along one face of the β-helix) and a C-terminal hydrophobic loop region (243-PFVPAPEVV-251). Unexpected

2012-01-01T00:00:00Z