• 한국미생물·생명공학회지
• /
• 제24권4호
• /
• pp.478-484
• /
• 1996

Recombinant human epidermal growth factor (rhEGF) was produced by E. coli BL21 harboring a plasmid pYHB101. The maximum production was 68.7 mg/l when the E. coli strain was cultured at 25$\circ$C for 48 hours in the modified MBL medium containing 10 g/l glucose with 1 mM IPTG induction at 2 hours after inoculation. The rhEGF was purified upto 267 folds by Amberlite XAD- 7 chromatography, ultrafiltration, and DEAE Sepharose fast flow ion exchange chromatography with an overall yield of 66.6%. The purified rhEGF was further separated into two fractions by HPLC. The N-terminal amino acid sequence of the second fraction was Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His. The effect of rhEGF on the DNA synthesis was examined using in vitro biological assay based on the incorporation of 5'-bromo-2'- deoxy-uridine (BrdU). The purified rhEGF shows no difference with natural human epidermal growth factor (nhEGF) in N-terminal amino acids residues and biological activity. From the results, we concluded that rhEGF produced from E. coli harboring the plasmid pYHB101 was apparently the same as nhEGF.

• Journal of Microbiology and Biotechnology
• /
• 제12권2호
• /
• pp.306-311
• /
• 2002

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationally modified compound in eukaryotic cells. Human O-GlcNAc transferase (OGT) was produced as a maltose binding protein (MBP) fusion protein, which showed significant catalytic activity to modify recombinant Sp1, transcription factor. To facilitate the production of O-GlcNAc modified proteins, instead of using the tedious in vitro glycosylation reaction or expression in eukaryotic cells, a MBP-fusion OGT expression vector (pACYC184-MBPOGT) was constructed using pACYC184 plasmid, which could coexist with general prokaryotic expression vectors containing ColE1 origin. By cotransforming pACYC184-MBPOGT and pGEX-2T vectors into Escherichia coli BL21, intracellular O- GlcNAcylated proteins could be obtained by a simple purification procedure. It is expected that this may be a useful tool for production of O-GlcNAc modified proteins.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권4호
• /
• pp.304-308
• /
• 2005

The response of IPTG induction was investigated through the monitoring of the alkali consumption rate and buffer capacity during the cultivation of recombinant E. coli BL21 (DE3) harboring the plasmid pRSET-LacZ under the control of lac promoter. The rate of alkali consumption increased along with cell growth, but declined suddenly after approximately 0.2 h of IPTG induction. The buffer capacity also declined after 0.9 h of IPTG induction. The profile of buffer capacity seems to correlate with the level of acetate production. The IPTG response was monitored only when introduced into the mid-exponential phase of bacterial cell growth. The minimum concentration of IPTG for induction, which was found out to be 0.1 mM, can also be monitored on-line and in-situ. Therefore, the on-line monitoring of alkali consumption rate and buffer capacity can be an indicator of the metabolic shift initiated by IPTG supplement, as well as for the physiological state of cell growth.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권3호
• /
• pp.225-229
• /
• 2005

To investigate impediments to plasmid transformation in Brevibacterium flavum BF4 and B. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties. B. lactofermentum was more sensitive to growth inhibition by glycine than B. flavum. Release of cellular proteins during sonication was more rapid for B. lactofermentum than for B. flavum. Plasmid DNA (pCSL 17) isolated from B. flavum transformed recipient $McrBC^+$ strains of Escherichia coli with lower efficiency than $McrBC^-$. McrBC digestion of this DNA confirmed that B. flavum contain methylated cytidines in the target sequence of McrBc sequences but B. lactofermentum contained a different methylation pattern. DNA derived from the B. lactofermentum transformed recipient $EcoKR^+$ strains of E. coli with lower efficiency than $EcoKR^-$, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.

• Journal of Microbiology and Biotechnology
• /
• 제14권4호
• /
• pp.777-782
• /
• 2004

The arylsulfatase gene (astA, 984 bp ORF) from the P. carrageenovora genome was amplified by PCR and subcloned into the pET21a vector. When the constructed plasmid pAST-A1 (6.4 kb) was introduced into E. coli BL21(DE3), the transformant on the LB plate containing IPTG showed a hydrolyzing activity for 4-methylumbelliferyl sulfate and p-nitrophenyl sulfate. The highest arylsulfatase activity (2.1 unit/ml) was obtained at 10 mM IPTG. Most arylsulfatase activity was found in the cell lysate, whereas no significant activity was detected in the culture supernatant. The molecular weight of the recombinant enzyme was estimated to be 33.1 kDa by SDS-PAGE. After the reaction of agar with arylsulfatase for 12 h at $40^{\circ}C$, the gel strength of the agar increased by 2-fold, and 73% of the sulfate in the agar had been removed. This result suggests that arylsulfatase expressed in E. coli could be useful in the production of electrophoretic grade agarose.

• Journal of Microbiology and Biotechnology
• /
• 제25권1호
• /
• pp.89-97
• /
• 2015

Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40℃, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (k_cat/K_m) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45℃, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176^th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca²⁺, whichincreased the thermostability of M179.

• 한국미생물·생명공학회지
• /
• 제26권1호
• /
• pp.15-22
• /
• 1998

일반적으로 Streptomyces류는 성장이 늦고 유지가 어려운 반면, E. coli는 배양기간이 짧고 유전자 조작도 간편한 장점이 있기 때문에, E. coli를 이용하여 유용단백질을 생산하는 연구가 일반적인 흐름이다. 그러나 E. coli에서 외래유전자를 도입하여 대량으로 생산을 시키는 경우에 비용해성의 inclusion body를 형성하는 경우가 많으므로 용해성의 활성형 단백질을 생산하기 위하여는 여러 가지 조건을 고려하여야 한다. 본 논문에서는 aklavlnone 11-hydroxylase gene(dnrF)을 E. coli BL2l에서 발현시킬 때의 배양조건을 배양온도와 IPTG농도의 두가지 요소를 조합하여 변형시키는 방법으로, 활성형 단백질의 생산을 최대화하고 inclusion body의 형성을 최소화하는 배양조건을 조사하였다. 그 결과, 37$^{\circ}C$에서 배양했을 때에는 0.02mM의 IPTG를 첨가하였을 때 inclusion body를 가장 적게 만들고, 그에 따라 생산되는 효소활성도 가장 높았다. 반면, 28$^{\circ}C$로 배양온도를 낮추었을 때에는 0.06mM의 IPTG를 첨가하였을 때 aklavinone 11-hydroxylase효소가 최대로 생산됨을 SDS-PAGE 및 효소 활성측정으로 확인하였다. IPTG농도를 0.1 mM로 높인 경우에는 28$^{\circ}C$, 37$^{\circ}C$에서 모두 aklavinone 11-hydroxylase효소가 과발현되어 Inclusion body를 가장 많이 생성하였음을 알 수 있었다. 방선균에서 동일 유전자를 대량발현시키는 경우 발현된 단백질의 효소 활성은 있으나 SDS-PAGE상에서의 단백질의 관찰이 불가능하였고, 동시에 단백질의 정제시 효소활성이 소실되어 정제가 불가능하였다. 이러한 효소 활성의 소실의 원인은 세포내의 어떤 저분자물질일 것으로 추정되며 본 연구에서 제작한 antibody를 이용하면, 효소의 정제가 용이하게 수행될 것이다. 특히 대장균계에서의 활성형 효소의 최적발현조건에서 세포를 배양하거나, inclusion body의 refolding을 실시한 후, 항체를 이용한 Western blot assay를 지표로 효소를 정제하면, 미지의 cofactor의 정체도 밝혀지고 aklavinone 11-hydroxylase류의 효소 특성 연구에 큰 도움이 될 것이다. 또한 이 효소를 이용한 다양한 종류의 bioconversion을 실시하여 그동안 background 때문에 생성된 product의 검출이 불가능했었던 소량의 생성산물의 분석도 가능할 것으로 판단되어 금후의 bioconversion연구에 기대하는 바가 크다.

• 대한의생명과학회지
• /
• 제8권3호
• /
• pp.107-113
• /
• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• 한국미생물·생명공학회지
• /
• 제48권4호
• /
• pp.480-490
• /
• 2020

A novel lipase gene, Lip-1420, was isolated from a metagenomic library constructed from reed marsh from Mt. Jumbong in Korea, comprising 112,500 members of recombinant plasmids. The DNA sequence of Lip-1420-subclone (5,513 bp) was found to contain at least 11 ORFs according to the GenBank database. The ORF-3 gene was inserted into the pET21a plasmid containing the C-terminal 6-His tag and transformed into E. coli BL21(DE3) to express the recombinant lipase protein. Lip-1420 was purified using a fast protein liquid chromatography system. The gene was registered in GenBank (MH628529). The values of Km and Vmax were determined as 0.268 mM and 1.821 units, respectively, at 40℃ and pH 8.0, using p-nitrophenyl palmitate as the substrate. This lipase belongs to family IV taxonomically because it has conserved HGGG and GDSAG motifs in the constitutive amino acid sequence. According to the predicted structural model, the binding sites are represented by residues H78, G81, D150, S151, A152, V181, and D236. Finally, Lip-1420 showed triolein selectivity for methanolysis between triolein (18:1) and tristearin (18:0) substrates. Further study of the selective mechanism and structure-function relationship of this new lipase could be useful for more practical applications.

• Journal of Microbiology and Biotechnology
• /
• 제13권2호
• /
• pp.292-300
• /
• 2003

Pseudomonas fluorescens B16 is a plant glowth-prornoting rhizobacterium, which produces an antibacterial compound that is effective against plant root pathogens, such as Agrobacrerium tumefaciens and Raistonia solanacearum. We mutagenized the strain B16 with Omegon-Km and isolated six antibacterial-activity-deficient mutants. Two cosmid clones that hybridized with the mutant clones also were isolated from a genomic library of tile parent strain. Using deletion and complementation analyses, it was found that the biosynthesis genes resided in a 4.3-kb SalI-NarI fragment. When a plasmid clone carrying the fragment was introduced into P. fluorescens strain 1855.344, which does not exhibit any antibacterial activity, the transconjugants exhibited antibacterial activity, indicating that the plasmid clone carried all the genes essential for production of the antibacterial compound. DNA sequence analysis of the fragment identified four putative open reading frames (ORFs): orf1 through orf4 The deduced amino acid sequences of ORF1, ORF2, and ORF4 were similar to cystathionine gamma lyase, pyruvate formate-lyase activating enzyme, and transcriptional regulator, respectively, yet the amino acid sequence of ORF3 showed no similarities to any known proteins. It was also demonstrated that the antibacterial activity was responsible for biological control of the bacterial wilt caused by R. solanacearum.