• Asian-Australasian Journal of Animal Sciences
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• 제17권12호
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• pp.1751-1757
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• 2004

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble $\beta$-galactosidase, the gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase (KNOUC112 $\beta$-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 $\beta$-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The $\beta$-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion $\beta$ -galactosidase expressed via pThioHis C at 37$^{\circ}C$ was about 5 times higher than that of unfused $\beta$-galactosidase expressed via pET-5b at 37$^{\circ}C$. Inclusion body of $\beta$-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 $\beta$ -gal was expressed via pET-5b at 37$^{\circ}C$. Fusion $\beta$ -galactosidase expressed at 37$^{\circ}C$ via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25$^{\circ}C$ prevented the formation of inclusion body, optimally at 25$^{\circ}C$. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25$^{\circ}C$. The soluble production of Thermus thermophilus KNOUC112 $\beta$-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

• BMB Reports
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• 제34권5호
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• pp.395-401
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• 2001

Schizosaccharomyces pombe gene encoding redox enzymes, such as thioltransferase (TTase) and thioredoxin (TRX), were previously cloned and induced by oxidative stress. In this investigation, their expressions were examined using $\beta$-galactosidase fusion plasmids. The expression of the two cloned genes appeared to be growth-dependent. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was increased in the medium with the low glucose level, whereas it was significantly decreased in the medium without glucose or with galactose. It was also decreased in the nitrogen-limited medium. The synthesis of galactosidase from the TRX-lacZ fusion was unaffected by galactose or low glucose. However, it was lowered the absence of glucose. The synthesis of $\beta$-galactosidase from the TTase-lacZ fusion was shown to be enhanced in a higher medium pH. Our findings indicate that S. pombe TTase and TRX genes may be regulated by carbon and nitrogen sources, as well as medium pH.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.663-669
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• 2000

The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with $\beta$-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur $\beta$-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.

• KSBB Journal
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• 제26권3호
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• pp.199-205
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• 2011

Using tetrameric ${\beta}$-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell ${\beta}$-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. ${\beta}$-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell ${\beta}$-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell ${\beta}$-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

• Journal of Microbiology and Biotechnology
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• 제1권4호
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• pp.274-280
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• 1991

Effects of IPTG induction on cell growth and production of ${\beta}$-galactosidase-preS2 fusion protein (${\beta}$gal-preS2) were studied in a defined medium using a recombinant Escherichia coli JM109/pCMHB30. IPTG was added (0.2 mM) to induce the cloned-gene expression in the early-, mid-, and late-log growth phases. The most serious decreases in growth rate and plasmid stability were observed for the induction in the early-log growth phase. The expression level of ${\beta}$gal-preS2 attained by the induction in the mid-log phase was about 0.51 mg fusion protein/mg total cellular protein, which was 2- and 5-fold improvement over the levels obtained with the inductions in the early- and late-log phases. Formation of acidic byproducts including acetate and pyruvate showed different profiles during the fermentation period for each cases of induction; pyruvate was the major byproduct for the induction in the early-log phase while acetate production became more significant for the cases of inductions in the mid- and late-log phases.

• KSBB Journal
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• 제10권1호
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• pp.38-45
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• 1995

Aspergillus oryzae에서 A. nidulans의 glyceral d dehyde-3-phosphate dehydrogenase (gpdA)와 trpC, prmoter의 발현 능력 을 E. coli lacZ gene fusion을 이용하여 비교.분석하였다. A. oryzae 내에서 발현된 E. coli $\beta$galactosidase의 specific activIty를 조사하여 본 결과, gpdA promoter를 가지는 transformant들 에서는 2,000unit/ug of protem 정도의 activity를 보이는 반면, trpC, promater를 가지고 있는 transformant들에서는 10.5~52.3unit/ug of protein 정도의 activity를 보였다. 이 결과로부터 A. oryzae 내에서 A. nidulans의 gpdA promoter가 trpC, promoter에 비해 70 배 정도 더 강한 발현 능력을 보이고 있음을 알 수 있다. Western blot 분석에서도 gpdA promoter를 가지고 있는 transf ormant에서 더 많은 E. coli $\beta$-galactosidase가 발현된 것 을 보여 주고 있다. 또한 southern blot 분석에서는 이러한 강한 발현이 transform된 plasmid의 copy number와 상관 없음을 보여주고 있다.

• 미생물학회지
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• 제30권5호
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• pp.371-376
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• 1992

플라스미드 pKM101 과 이의 돌연변이체 pSL4 는 UV 및 MMS 에 대해 높은 치사 저항성과 돌연변이률을 나타낸다. pKM101 과 pSLA 의 mucB 유전자의 일부분과 mucA 유전자를 lacZ fusion 벡터인 pMC874 에 subcloning 시켜 플라스미드 pBH31 과 pBH30 을 선별하였고 이 플라스미드들을 $recA^{+}lexA^{-}$, $recA^{-}와lexA^{+}$, 균주에 각각 도입하였다. $recA^{+}lexA^{+}$ 균주에서 pSLA 의 muc 유전자를 포함하는 pBH30 의 $\beta$-galactosidase활성은 pKM101 의 muc 유전자가 연결된 pBH31 보다 높았고 $recA^{-}$ 와 $lexA^{-}$ 돌연변이주에서는 UV 조사의 유무에 관계없이 $\beta$-galactosidase를 유도하지 못하였지만 $recA^{-}$ 균주에서는 UV 조사를 하지 않았을 때 pBH30 의 $\beta$-galactosidase가 pBH31 보다 약간 높게 유도되었다. 이러한 결과는 pKM101 과 pSLA 의 기능적 차이가 두 플라스미드의 Muc 단백질의 구조적 차이라고 생삭할 수 있지만, muc 유전자의 조절부위가 돌연변이되너 LexA repessor 가 작용하는 부위의 변화에 의한 것이라고도 생각햐ㄹ 수 있었다. 또한, umuC-lacZ 유전자를 가지는 pBH100 를 construction 하여 umu oeron 의 발현은 UV 조사에 의해 유도되며 recA 와 lexA 유전자에 의해 조절됨을 알 수 있었다.

• 미생물학회지
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• 제32권1호
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• pp.12-19
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• 1994

Saccharomyces cerevisiae의 KEMI 유전자는 세포의 영양 상태에 따라 spindle pole body나 microtubules의 기능을 조절하는 것으로 알려져 있다. 이 유전자 산물의 세포내 분포 및 기능을 규명하기 위하여, KEM1::lacZ 융합 유전자를 제조하였다. 즉, 클론된 KEM1 유전자에 대장균의 ${\beta}$-galactosidase 구조유전자를 갖는 mini-Tn10-LUK element를 무작위 삽입한 pool을 제조하고, 이를 분석하여 KEM1의 기능 부위가 약 3.5kb에 해당함을 확인하였고, KEM1의 기능이 살아있는 KEM1::lacZ 융합 유전자의 클론을 선별하였다. 이 클론을 ${\beta}$-galactosidase 항체를 이용한 indirect immunofluorescence 방법으로 분석하여 KEM1::lacZ 융합 단백질이 핵주변에 위치함을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제1권3호
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• pp.151-156
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• 1991

In order to construct a yeast strain having high ethanol tolerance together with good lactose fermentation ability, the protoplast fusion using Saccharomyces cerevisiae STV 89 and Kluyveromyces fragilis CBS 397 was carried out. Auxotrophic mutants of K. fragilis were obtained as a selection marker by treatment of ethylmethane sulfonate. The best mutant for protoplast fusion was selected based on the capabilities of ${\beta}-galactosidase$ production and lactose fermentation. The protoplast fusion using polyethylene glycol and calcium chloride solution led to the fusion frequence of $3{\times}10^{-6}$ and a number of fusants were obtained. Among these fusants, a fusant F-3-19 showed the best results in terms of ethanol tolerance, ${\beta}-galactosidase$ activity and lactose fermentation. The performance of lactose fermentation and ethanol tolerance by this fusant were better than those of K. fragilis. Study on the ethanol tolerance having relation to fatty acid composition and intracellular ethanol concentration revealed that the fusant F-3-19 had a higher unsaturated fatty acids content and accumulated less amount of intracellular ethanol compared with a parent of K. fragilis.

• Molecules and Cells
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• 제20권1호
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• pp.43-50
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• 2005

Glutaredoxin (Grx) is a small, heat-stable redox protein acting as a multi-functional glutathione (GSH)-dependent disulfide oxidoreductase. We have cloned the monothiol Grx5 gene from the genomic DNA of the fission yeast Schizosaccharomyces pombe. It has 1,904 bp, with one intron, and encodes a putative protein of 146 amino acids with a molecular mass of 16.5 kDa. Recombinant Grx5 produced functional Grx in S. pombe cells. NO-generating sodium nitroprusside (SNP, 1.0 and 2.0 mM) and potassium chloride (KCl, 0.2 and 0.5 M) increased the synthesis of ${\beta}$-galactosidase from a Grx5-lacZ fusion gene, and transcription of Grx5 was also enhanced by SNP and KCl. Synthesis of ${\beta}$-galactosidase from the Grx5-lacZ fusion was lower in Pap1-negative TP108-3C cells than in wild type KP1 cells, and when Pap1 was overproduced in KP1 cells, the level of ${\beta}$-galactosidase increased. We also found that Pap1 is involved in the induction of Grx5 by SNP and KCl. S. pombe Grx5 may play a crucial role in responses to nitrosative and osmotic stresses.