• Molecules and Cells
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• 제27권4호
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• pp.475-480
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• 2009

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.

• 한국미생물·생명공학회지
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• 제49권1호
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• pp.75-87
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• 2021

Type I Interferons (IFNα) are known for their role as biological anticancer agents owing to their cell-apoptosis inducing properties. Development of an appropriate, cost-effective host expression system is crucial for meeting the increasing demand for proteins. Therefore, this study aims to develop codon-optimized IFNα-2b in L. lactis NZ3900. These cells express extracellular protein using the NICE system and Usp₄₅ signal peptide. To validate the mature form of the expressed protein, the recombinant IFNα-2b was screened in a human colorectal cancer cell line using the cytotoxicity assay. The IFNα-2b was successfully cloned into the pNZ8148 vector, thereby generating recombinant L. lactis pNZ8148-SP_Usp45-IFNα-2b. The computational analysis of codon-optimized IFNα-2b revealed no mutation and amino acid changes; additionally, the codon-optimized IFNα-2b showed 100% similarity with native human IFNα-2b, in the BLAST analysis. The partial size exclusion chromatography (SEC) of extracellular protein yielded a 19 kDa protein, which was further confirmed by its positive binding to anti-IFNα-2b in the western blot analysis. The crude protein and SEC-purified partial fraction showed IC₅₀ values of 33.22 ㎍/ml and 127.2 ㎍/ml, respectively, which indicated better activity than the metabolites of L. lactis NZ3900 (231.8 ㎍/ml). These values were also comparable with those of the regular anticancer drug tamoxifen (105.5 ㎍/ml). These results demonstrated L. lactis as a promising host system that functions by utilizing the pNZ8148 NICE system. Meanwhile, codon-optimized usage of the inserted gene increased the optimal protein expression levels, which could be beneficial for its large-scale production. Taken together, the recombinant L. lactis IFNα-2b is a potential alternative treatment for colorectal cancer. Furthermore, its activity was analyzed in the WiDr cell line, to assess its colorectal anticancer activities in vivo.

• 미생물학회지
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• 제52권4호
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• pp.455-462
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• 2016

Pseudomonas aeruginosa P-5 균주는 홀수개의 탄소수를 갖는 지방산으로부터 3-hydroxyvalerate (3HV)와 medium-chain-length (MCL) 3-hydroxyalkanoates (3HAs) 단위체로 구성된 popolyhydroxyalkanoate (PHA) 공중합체를 생산하는 특이한 성질을 갖고 있다. 이 균주가 갖고 있는 2개의 MCL-PHA synthases ($PhaC1_{P-5}$와 $PhaC2_{P-5}$)의 탄소길이에 따른 기질특이성을 비교하기 위하여 각각의 유전자를 PHA 생합성능이 결여된 돌연변이주 Pseudomonas putida GPp104에 도입하고 발현시킨 결과, $PhaC2_{P-5}$는 3HV와 MCL 3HAs로 이루어진 공중합체를 생산하지만 $PhaC1_{P-5}$는 단지 MCL 3HAs로 구성된 공중합체를 생산하였다. 이는 $PhaC2_{P-5}$가 $PhaC1_{P-5}$과는 달리 보다 짧은 탄소길이의 3-hydroxyvaleryl Co-A를 기질로 인지하여 합성반응에 이용할 수 있음을 보여주는 것이다. 또한 $PhaC2_{P-5}$의 효소활성 및 기질특이성의 변화를 유도하기 위하여 위치지정 돌연변이생성을 수행하고 P. putida GPp104과 다른 PHA 생합성능 결여 돌연변이주인 Ralstonia eutropha $PHB^-4$에서 발현시킨 결과, $PhaC2_{P-5}$ 내 두 개 아미노산의 치환(Ser326Thr과 Gln482Lys)이 공중합체의 3HV 함량을 크게 증진시키는 효과를 보였다. 두 개 아미노산이 모두 치환된 $PhaC2_{P-5}$ 유전자($phaC2_{P-5}QKST$)를 갖는 P. putida GPp104를 nonanoic acid가 탄소원으로 함유된 배지에서 배양하였을 때, 모균주에 비해 공중합체 함량과 공중합체 내 3HV 함량이 각각 2.5배 및 3.5배 증가하였다. 따라서 $phaC2_{P-5}QKST$를 포함하는 재조합 균주는 개량된 물성의 신규PHAs 생산에 유용할 것으로 기대된다.