• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.545-554
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• 2013

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

• KSBB Journal
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• 제23권3호
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• pp.231-238
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• 2008

암세포의 침윤은 숙주 조직의의 기저막과 세포외 기질을 침투함으로써 일어난다. 침윤과 전이과정에는 단백질가수분해 효소인 matrix metalloproteinases (MMPs)가 깊이 연관되어 있는 것으로 알려져 있으며, MMP의 가수분해 활성은 tissue inhibitors of metalloproteinases (TIMPs)라는 억제 단백질에 의해 억제된다. TIMP-2는 21kDa 크기의 포유류 단백질로 대장균에서 과발현 시 다른 많은 포유류 단백질과 마찬가지로 가용성이 낮은 봉입체 형태로 발현된다. TIMP-2 단백질의 접힘에 6개의 이황화결합이 필요하고, 이는 일반적으로 대장균 환경은 적합하지 않다. 본 연구에서는 대장균에서 불가용성으로 발현되는 TIMP-2 유전자를 유전자셔플링 기법의 한 가지인 StEP (staggered extension process)를 변형하고 동시에 $Mn^{2+}$ 농도 변화와 dGTP 불균형을 이용한 무작위 돌연변이 기법을 혼용하여 대장균에서 가용성 TMP-2 재조합 변이체를 생성하고자 하였다. 무작위로 재조합된 TIMP-2 유전자 중에서 가용성으로 발현되는 TIMP-2 유전자를 선별하기 위해서 chloramphenicol acetyltransferase (CAT)-융합 방법을 도입하였다. CAT 유전자가 가용성으로 발현되는 재조합 TIMP-2 유전자에 융합되면 이를 갖는 E. coli는 높은 chloramphenicol 환경에서 생존이 가능하게 된다. 이러한 in virro mutageuesis 기법과 CAT-융합 방법으로 대장균 가용성 TIMP-2 재조합 변이체를 14가지 얻을 수 있었다. 변이체 TIMP-2의 아미노산서열 분석과 구조 분석 결과 주로 소수성 아미노산이 친수성 아미노산으로 전환되었고, MMP와의 결합이 관여하지 않는 C-말단 부위에 돌연변이가 집중되어 있었다. 본 연구에서 개발된 간편하고 새로운 in vitro 재조합 방법과 CAT을 이용한 스크리닝 기법은 다른 많은 대장균 내 불가용성 단백질의 발현에도 사용될 수 있을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제32권9호
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• pp.1430-1438
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• 2019

Objective: This experiment was designed to determine the effects of coated cysteamine hydrochloride (CC) on muscle fiber characteristics, amino acid composition and transporters gene expression in the longissimus dorsi muscle (LDM) of finishing pigs. Methods: Two hundred and sixteen Duroc/Landrace/Yorkshire cross-bred male finishing pigs were fed with a corn-soybean basal diet supplemented with 0, 70, and 140 mg/kg cysteamine. Each group contained eight replicates of nine pigs per replicate. After 29 days, one pig was randomly selected from each replicate and slaughtered. Blood and LDM samples were collected and analyzed. Results: The results showed that supplemental dietary CC increased (p<0.05) the muscle fiber density. And CC supplementation also up-regulated (p<0.05) the expression of myosin heavy chain 1 (MyHC1) and MyHC2x mRNA levels, and down-regulated (p<0.05) MyHC2b expression in the LDM. Additionally, supplemental dietary CC reduced (p<0.05) the concentration of total cholesterol in the plasma and enhanced (p<0.05) the concentrations of essential amino acid and total amino acid in the LDM. The relative expression levels of chloramphenicol acetyltransferase 2, $b^{0,+}$ amino acid transporter, and $y^+$-L-type amino acid transporter 1 were upregulated (p<0.05) in the LDM when pigs were fed with the dietary CC of 70 mg/kg. Conclusion: Cysteamine supplementation could increase fiber density and distribution of fiber types. It also improved the deposition of protein in the LDM by up-regulated the expression of amino acid transporters.