• 생명과학회지
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• 제27권5호
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• pp.584-590
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• 2017

결빙방지 단백질(Antifreeze protein, AFP)은 얼음 결정에 결합하여 결정의 성장을 억제한다. AFP는 영하의 환경에서 서식하는 생물체의 생존에 필수적이다. 겨울 넙치에서 분리된 type I AFP (AFP37)는 37 개의 잔기를 가진 ${\alpha}$-나선 구조의 펩타이드이다. 본 연구에서는 활성과 수용성이 높은 짧은 AFP 조각을 개발하고자 시도하였다. 아미노-말단 15, 21 잔기의 AFP15와 21를 설계 및 합성하였다. 이들 펩타이드의 아미노-말단에 CGG를 도입한 AFP15N and 21N을 합성하고 이황화 결합을 유도함으로써 이량체 펩타이드인dAFP15N과dAFP21N을 합성하였다. 이들의 나선 함량과 결빙방지 활성을 circular dichroism (CD) 분광법과 나노리터 삼투압계로 각각 측정하였다. 합성된 펩타이드 AFP15 AFP21, AFP15N, AFP21N, dAFP15N, dAFP21N의 나선 구조 함량은 대조구인 AFP37의 49, 41, 23.8, 28, 47.9, 51.7% 수준을 보였다. 이들의 결빙방지 활성은 AFP37의 13, 7, 0.05, 5.6, 13, 11%로 나타났다. 예상과는 달리 이량체화된 펩타이드는 단량체와 비슷한 결빙방지 활성을 보였다. 이는 이량체 펩타이드가 하나의 펩타이드로 얼음과 결합하기 보다 두 개의 개별적 펩타이드로 작용함으로써 단량체와 같은 활성을 보인 것으로 생각된다. 또한 펩타이드들에 의한 별 모양의 얼음 결정 형성은 펩타이드와 얼음의 약한 결합을 시사한다.

• Clinical and Experimental Reproductive Medicine
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• 제44권1호
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• pp.8-14
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• 2017

Objective: The aim of this study was to analyze the effect of supplementing vitrification and warming solutions with two types of antifreeze proteins (AFPs) and the combination thereof on the follicular integrity of vitrified-warmed mouse ovaries. Methods: Ovaries (n=154) were obtained from 5-week-old BDF1 female mice (n=77) and vitrified using ethylene glycol and dimethyl sulfoxide with the supplementation of 10 mg/mL of Flavobacterium frigoris ice-binding protein (FfIBP), 10 mg/mL of type III AFP, or the combination thereof. Ovarian sections were examined by light microscopy after hematoxylin and eosin staining, and follicular intactness was assessed as a whole and according to the type of follicle. Apoptosis within the follicles as a whole was detected by a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Results: The proportion of overall intact follicles was significantly higher in the type III AFP-supplemented group (60.5%) and the combination group (62.9%) than in the non-supplemented controls (43.8%, p<0.05 for each). The proportion of intact primordial follicles was significantly higher in the FfIBP-supplemented (90.0%), type III AFP-supplemented (92.3%), and combination (89.7%) groups than in the non-supplemented control group (46.2%, p<0.05 for each). The proportions of non-apoptotic follicles were similar across the four groups. Conclusion: Supplementation of the vitrification and warming solutions with FfIBP, type III AFP, or the combination thereof was equally beneficial for the preservation of primordial follicles in vitrified mouse ovaries.

• Fisheries and Aquatic Sciences
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• 제19권8호
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• pp.33.1-33.7
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• 2016

Antifreeze proteins (AFPs) lower the freezing point but not the melting point of aqueous solutions by inhibiting the growth of ice crystals via an adsorption-inhibition mechanism. However, the function of type IV AFP (AFP IV) is questionable, as its antifreeze activity is on the verge of detectable limits, its physiological concentration in adult fish blood is too low to function as a biological antifreeze, and its homologues are present even in fish from tropic oceans as well as freshwater. Therefore, we speculated that AFP IV may have gained antifreeze activity not by selective pressure but by chance. To test this hypothesis, we cloned, expressed, and assayed AFP IV from cultured subtropical olive flounder (Paralichthys olivaceus), which do not require antifreeze protein for survival. Among the identified expressed sequence tags of the flounder liver sample, a 5'-deleted complementary DNA (cDNA) sequence similar to the afp4 gene of the longhorn sculpin was identified, and its full-length cDNA and genome structure were examined. The deduced amino acid sequence of flounder AFP IV shared 55, 53, 52, and 49 % identity with those of Pleuragramma antarcticum, Myoxocephalus octodecemspinosus, Myoxocephalus scorpius, and Notothenia coriiceps, respectively. Furthermore, the genomic structure of this gene was conserved with those of other known AFP IVs. Notably, the recombinant AFP IV showed a weak but distinct thermal hysteresis of $0.07{\pm}0.01^{\circ}C$ at the concentration of 0.5 mg/mL, and ice crystals in an AFP IV solution grew star-shaped, which are very similar to those obtained from other polar AFP IVs. Taken together, our results do not support the hypothesis of evolution of AFP IV by selective pressure, suggesting that the antifreeze activity of AFP IV may have been gained by chance.

• KSBB Journal
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• 제30권6호
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• pp.345-349
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• 2015

Antifreeze proteins (AFP) inhibit growth and recrystallization of ice, and permit organisms to survive in cold environments. The AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris icebinding protein), was produced in E. coli using a cold shock induction system. The culture temperature was shifted from $37^{\circ}C$ to $15^{\circ}C$ and a 20 L culture scale was used. The final weights of dried cell and FfIBP were estimated to be 126 g and 8.4 g, respectively. The thermal hysteresis (TH) activity ($1.53^{\circ}C$) of the produced FfIBP was 3.6-fold higher than that of the LeIBP (Leucosporidium ice-binding protein) produced in Picha. The current study demonstrates that large-scale production of FfIBP was successful and the result could be extended to further application studies using recombinant AFPs.

• KSBB Journal
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• 제29권4호
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• pp.303-306
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• 2014

Antifreeze proteins (AFP) inhibit ice growth to permit the survival of polar organisms in the cold environments. The recombinant AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris ice-binding protein), was produced using Pichia pastoris expression system. The optimum fermentation temperature ($30^{\circ}C$) and pH (5) for FfIBP production were determined using a fed-batch culture system. The maximal cell density and purified FfIBP were 112 g/L and 70 mg/L, respectively. The thermal hysteresis (TH) activity (0.85) of FfIBP obtained using a glycerol-methanol fed-batch culture system was 2-fold higher than that of the LeIBP (Leucosporidium ice-binding protein). This work allows for large-scale production of FfIBP, which could be extended to further application studies using recombinant AFPs.