• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1076-1080
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• 2008

Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat ${\alpha}$-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.

• 유기물자원화
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• 제11권3호
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• pp.67-74
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• 2003

본 연구의 목적은 토양미생물의 일종인 Pseudomonas putida 에서 추출한 탄소고갈 유전자로부터 starvation promoter를 분리한 후 starvation vector를 개발하여 이를 미생물로부터 유용물질의 생산 및 오염물질의 정화등에 활용하기 위한 것이다. Starvation 유전자란 영양분의 결핍시에만 특수하게 발현되는 유전자의 일종으로 본 유전자로부터 promoter를 분리한 후 특정 물질을 분해 또는 생성하는 유전자를 분리된 promoter에 유전자 재조합 방법에 의하여 연결시키면 영양분의 공급을 최소화하고 세균의 생장을 억제 시키는 동시에 promoter에 연결된 유전자의 발현 기능은 최대화 시킴으로써 그 목적을 달성할 수 있다 하겠다. 본 연구에서는 기 분리된 starvation gene으로부터 promoter를 분리한 후 이를 적절한 벡터에 연결시켜 starvation vector인 pYKS101을 완성하였다. 본 벡터의 성능을 시험하기 위하여 reporter gene으로 lacZ유전자를 벡터내에 클로닝하여 세균의 염색체에 삽입시킨후 그 성능을 조사하였다. 삽입된 유전자는 예상한 바와같이 세균이 영양분이 결핍되었을 때 충분한 영양분이 공급되었을 때보다 약 4배의 활성도를 나타냄으로써 본 벡터의 유용성을 입증할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1125-1129
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• 2005

Cloning of a 6.6-kb BamHI digested chromosomal DNA from S. griseus IFO13350 revealed the presence of an additional gene encoding a novel trypsin-like enzyme, named SprU. The SprU protein shows a high homology ($79\%$ identity, $88\%$ similarity) with the SGT protease, which has been reported as a bacterial trypsin in the same strain. The amino acid sequence deduced from the nucleotide sequence of the sprU gene suggests that SprU is produced as a precursor consisting of an amino-terminal presequence (29 amino acid residues), prosequence (4 residues), and mature trypsin consisting of 222 amino acids with a molecular weight of 22.94 kDa and a calculated pI of 4.13. The serine, histidine, and aspartic acid residues composing the catalytic triad of typical serine proteases are also well conserved. When the trypsin activity of the SprU was spectrophotometrically measured by the enzymatic hydrolysis of the artificial chromogenic substrate, N-${alpha}$-benzoyl-DL-arginine-p-nitroanilide, the S. lividans transformant with pWHM3-U gave 3 times higher activity than that of control. When the same recombinant plasmid was introduced into S. griseus, however, the gene dosage effect was not so significant, as in the cases of other genes encoding serine proteases, such as sprA, sprB, and sprD. Although two trypsins, SprU and SGT, have a high degree of homology, the pI values, the gene dosage effect in S. griseus, and the gene arrangement adjacent to the two genes are very different, suggesting that the biochemical and biological function of the SprU might be quite different from that of the SGT.

• Current Research on Agriculture and Life Sciences
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• 제17권
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• pp.59-63
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• 1999

벼의 엽록체 small HSP의 고온 스트레스 하에서의 기능을 밝히기 위하여 Oshsp21 cDNA를 pET 발현 vector에 도입하였다. 형질전환된 대장균 배양액에 IPTG를 첨가하여 단백질 발현을 유도시킨 다음 고온 stress 하에서의 생존율을 대조구와 비교하였다. 그 결과 대조균주의 경우 $50^{\circ}C$에서의 생존율이 크게 감소하였으나 Oshsp21이 발현된 대장균의 경우 70% 이상의 생존율을 나타내었다. 또한 대장균 단백질을 $55^{\circ}C$에서 30분간 열처리한 후, 대장균 단백질을 가용성과 비가용성 단백질로 분획한 다음, 각각의 비율을 조사한 결과, 대조균주의 경우 총 단백질의 약 60%가 비가용성 단백질로 변성되었으나, Oshsp21을 발현시킨 대장균의 경우 총 단백질의 약 35%만이 비가용성 단백질로 나타났다. 이러한 결과는 벼의 엽록체 small HSP는 세포내에서 분자 chaperone으로 기능하여 고온내성을 부여할 수 있음을 나타낸다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권6호
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• pp.850-855
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• 2007

Leptin is the adipocyte-specific product of the obese gene and plays a major role in food intake and energy metabolism. Leptin research was mainly focused on mammalian species, but understanding of leptin and its function in poultry is very poor. In this study, the duck leptin gene was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) from duck liver RNA. The cDNA fragment was inserted into the pET-28a expression vector, and the resulting plasmid was expressed in Escherichia coli BL21 (DE3). Experimental mice were given an intraperitoneal injection of 10 mg/kg leptin dissolved in phosphate buffered saline (PBS), while the control mice were injected with PBS. The effect of leptin on food intake, body weight and fatty deposition in mice was detected. Sequence analysis revealed that duck leptin had a length of 438 nucleotides which encoded a peptide with 146 amino acid residues. The sequence shares highly homology to other animals. The coding sequence of duck leptin was 84 and 86% identical to human and pig leptin nucleotides sequence. Highest identity was with the rat coding sequence (95%). The identity of the amino acid sequence was 84, 82 and 96% respectively compared to that of the human, pig and rat. Results of SDS-PAGE analysis indicated that a fusion protein was specifically expressed in E. coli BL21 (DE3). The purified product was found to be biologically active during tests. Continuous administration of recombinant duck leptin inhibited food intake. Despite the decrease of food intake, leptin significantly induced body weight and fatty deposition. These changes were accompanied by a significant down-secretion of plasma glucose, cholesterol, triglyceride and insulin levels in mice. The observations provide evidence for an inhibitory effect of leptin in the regulation of food intake and for a potential role of duck leptin in the regulation of lipogenesis.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1868-1874
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• 2007

We have developed a robotic system for an automated parallel cell cultivation process that enables screening of induction parameters for the soluble expression of recombinant protein. The system is designed for parallelized and simultaneous cultivation of up to 24 different types of cells or a single type of cell at 24 different conditions. Twenty-four culture vessels of about 200 ml are arranged in four columns${\times}$six rows. The system is equipped with four independent thermostated waterbaths, each of which accommodates six culture vessels. A two-channel liquid handler is attached in order to distribute medium from the reservoir to the culture vessels, to transfer seed or other reagents, and to take an aliquot from the growing cells. Cells in each vessel are agitated and aerated by sparging filtered air. We tested the system by growing Escherichia coli BL21(DE3) cells harboring a plasmid for a model protein, and used it in optimizing protein expression conditions by varying the induction temperature and the inducer concentration. The results revealed the usefulness of our custom-made cell cultivation robot in screening optimal conditions for the expression of soluble proteins.

• 미생물학회지
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• 제52권4호
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• pp.495-499
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• 2016

본 연구에서는 발광 세균에 존재하는 LuxG 단백질의 효소학적 성질을 알아내기 위하여 Photobacterium leiognathi ATCC 25521의 luxG 유전자를 중합효소연쇄반응으로 증폭시켜 T5 프로모터와 6X His-tag 시스템을 지닌 pQE 벡터에 삽입시킨 재조합플라스미드를 제조하여 대장균에 형질전환 후 과발현시켜 단백질을 분리, 정제 하였다. 정제된 단백질의 효소학적 실험 결과, 이 단백질은 FMN과 NADPH 기질에 대한 ferric iron reductase의 기능을 갖고 있음을 확인하였으며 이들 기질에 대한 효소 활성도 상수 $K_m$ 및 $V_{max}$ 값을 결정하였다.

• 미생물학회지
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• 제30권6호
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• pp.466-471
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• 1992

HTLV-I 의 gag-pro 유전자 중첩영역내에서 -1 ribosomal frameshifting 이 일어나는 자리를 결정하기 위하여 gag-pro 중첩영역의 일부를 SP6 promoter 를 가진 백터내에 클로닝하였다. 그 결과 닭의 prelysozyme 에서 유래한 5개의 아미노산을 코드하는 합성유전자와 141 bp 로된 gag-pro 중첩영역의 뒤에 Straphylococcus aureus 의 protein A 유전자단편이 연결된 hybrid 유전자를 보유한 플라스미드를 제작하였다. 이 DNA 클론을 주형으로 SP6 RNA polymerase 의 작용에 의해 한종류의 mRNA 를 다량으로 합성하였다. Invitro 에서 합성된 mRNA 로 무세포계에서 단백질을 합성한 결과 21 kDal 의 단백질이 생성되었고 IgG-Sepharose 를 사용한 affinity chromatography 로 합성된 단백질을 순수하게 정제할 수 있었다. 본연구에서 설명한 in vitro 실험계는 Gag-Pro transframe 단백질의 신속한 정제 및 일차구조의 결정에 유익하게 사용될 것으로 보이며 이와 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 같은 실험의 결과 mRNA 에서 ribosomal frameshifting 이 일어나는 정확한 site 를 결정할 수 있을 뿐 아니가 pro 유전자의 발현에 필요한 frameshift 를 유도하는 tRNA 의 동정도 가능하게 될 것이다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.644-648
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• 2005

Dextranase (${\alpha}$-1,6-D-glucan-6-glucanogydrolase:E.C. 3.2.1.11) catalyzes the hydrolysis of ${\alpha}$-(1.6) linkages of dextran. A lsd1 gene encoding an extracellular dextranase was isolated from the genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase (lsd1) after codon optimization for gene expression with Pichia pastoris system. A open reading frame of lsd11 gene was 1827 bp and it was inserted into the pPIC3.5K expression vector. The plasmid linearized by Sac I was integrated into the 5'AOX region of the chromosomal DNA of P. pastoris. The lsd11 gene fragment encoding a mature protein of 608 amino acids with a predicted molecular weight of 70 kDa, was expressed in the methylotrophic yeast P. pastoris by controling the alcohol oxidase-1 (AOX1) promoter. The recombinant lds11 was optimized by using the shake-flask expression and upscaled using fermentation technology. More than 9.8 mg/L of active dextranase was obtained after induction by methanol. The optimum pH of LSD11 was found to be 5.5 and the optimum temperature $28^{\circ}C$.

• BMB Reports
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• 제35권4호
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• pp.432-436
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• 2002

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XyITE products catalyze the dioxygenation of the aromatics. The sylT of the strain S-47 is located just upstream of the xylE gene. XylT of the strain S-47 is located just upstream of the xylE gene. XyIT is typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2(TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XyIT for the reactivation of the catechol 2,3-dioxygenase (XyIE) that is oxidized with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 mimutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XyIT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.