• Archives of Pharmacal Research
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• v.27 no.7
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• pp.776-780
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• 2004

Interferons are cytokines that confer resistance to viral infection and inhibit cellular proliferation. The interferon alpha gene from human blood samples was amplified, cloned and expressed in E. coli (BL21). Leukocyte chromosomal DNA was used as a source of template DNA. Using specific primers, the gene for HulFN$\{alpha}$-2b was amplified and inserted into the E. coli vector, pET21b, by ligation of the HindIII and BamHI linkers of the vector and insert. The insert was further analyzed by PCR, DNA restriction mapping and sequencing, and expressed in a suitable E. coli strain. The production of this important cellular protein in the laboratory has significant applications in production of the recombinant pharmaceutical proteins.

• Journal of Life Science
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• v.14 no.2
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• pp.315-319
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• 2004

For antibody development of human serine palmitoyltransferase (SPT, EC 2.3.1.50), SPTLC1 and SPTLC2 genes were subcloned in pRset vector and expressed in E. coli BL21 (DE3)pLys cells. Eucaryotic SPT is a membrane-bound heterodimer enzyme, while all other members are soluble homodimer enzymes. cDNA library were obtained from total RNA from human embryo kidney cell line, HEK293, using RT-PCR and PCR with specific primers was carried out for preparing SPTLC1 and SPTLC2 genes. pRset vector which can express hexahistidine-tag fusion protein was used and the DNA sequences of pRsetB/SPTLC1 and pRsetA/SPTLC2 were confirmed. Recombinant BL21 cells with SPTLC subunits were selected with LB plate containing ampicillin and chroramphenicol. SPTLC1 and SPTLC2 proteins were induced with 1 mM IPTG and seperated on 10% SDS-PAGE gel. Expressed proteins were confirmed by western blotting with His-tag antibody.

• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.89-97
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• 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.

• The Plant Pathology Journal
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• v.21 no.4
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• pp.334-342
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• 2005

The prototype Chlorella virus PBCV-l encodes 11 tRNA genes and over 350 protein-encoding genes in its 330 kbp genome. Initial attempts to overexpress the recombinant A189/192R protein, a putative virus attachment protein, in E. coli strain BL21(DE3) SI were unsuccessful, and multiple protein bands were detected on Western blots. However, the full-length A189/192R recombinant protein or fragments derived from it were detected when they were expressed in E. coli BL21 CodonPlus (DE3) RIL, which contains extra tRNAs. Codon usage analysis of the a189/192r gene showed highly biased usage of the AGA and AVA codons compared to genes encoded by E. coli and Chlorella. In addition, there were biases of XXA/U($56\%$) and XXG/ C($44\%$) in the codons recognized by the viral tRNAs, which correspond to the codon usage bias in the PBCV-1 genome of XXA/U ($63\%$) over those ending in XXC/G ($37\%$). Analysis of the codon usage in the major capsid protein and DNA polymerase showed preferential usage of codons that can be recognized by the viral tRNAs. The Asn (AAC) and Lys (AAG) codons whose corresponding tRNA genes are duplicated in the tRNA gene cluster were the most abundant (i.e., preferred) codons in these two proteins. The tRNA genes encoded in the PBCV-l genome seem to play a very important role during the synthesis of viral proteins through supplementing the tRNAs that are frequently used in viral proteins, but are rare in the host cells. In addition, these tRNAs would help the virus to adapt to a wide range of hosts by providing tRNAs that are rare in the host cells.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.575-578
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• 2000

The N-acetylneuraminate lyase(NALase) from Escherichia coli was cloned and it was compared to that from Haemophilus influenzae and Clostridium perfringens. NALase from E. coli was expressed in very high level(about 6U/mg). The ManNAc Km value of three enzymes was almost the same. Pyruvate inhibited from H. influenzae was inhibited by GlcNAc in lower level than the others. The crude extract has about 30 times more activity than the cell for the substrate and product diffusion limit problem. The pH stability of three enzymes at pH 11 was also checked for its importance in the direct synthesis of Neu5Ac from GlcNAc and pyruvate at high alkaline condition.

• KSBB Journal
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• v.13 no.6
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• pp.662-668
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• 1998

Viruses belonging to the Hantavirus genus cause two acute severe illness in humans, i.e., Haemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome(HPS). Among them, Hantaan virus is one of the most important viruses causing HFRS. Recombinant expression vectors, pKK-NP and pET-NP, with Hantaan viral nucleocapsid gene were constructed, and used to transform Eschericia coli BL21(DE3). Stability of the vectors in the host strain, and effects of some environmental conditions on the expression of nucleocapsid gene were studied. Expression vector, pKK-NP, was very unstable, and the expression level of nucleocapsid gene was very low compared to that of pET-NP. BL21(pET-NP) produced about 100 mg of N protein per liter of culture broth. Induction time did not show any significant difference on the expression level of nucleocapsid gen and cell growth. BL21(pET-NP) culture at 35$^{\circ}C$ showed a little higher expression level than at 30$^{\circ}C$ during growth phase, but reached to the same level at stationary phase. Total expression level was proportional to supplemented glucose concentration of media up to 0.5% along with cell growth, but expression level per unit cell mass was inversely proportional to glucose concentration and maximal when glucose was not supplemented at all.

• Biotechnology and Bioprocess Engineering:BBE
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• v.4 no.1
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• pp.63-65
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• 1999

Although many pharmaceutically useful proteins are produced in E. coli expression system, it is very are rare for the system to be used in the production of diagnostic antigen due to a major problem, i.e., false-positive reaction of e. coli host-derived proteins contaminating purified diagnostic antigen with human sera. The N (nucleocapsid) protein of Seoul virus causing haemorrhagic fever with renal syndrome (HFRS) was produced in E. coli BL21 (DE3), and used for the detection of N protein-specific antibodies in human sera. Using the N protein as a diagnostic antigen of HFRS, the false-positive reaction was cleared by merely mixing the test sera with the extract of E. coli host strain not harboring expression plasmid.

• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.777-782
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• 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.

• Current Research on Agriculture and Life Sciences
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• v.17
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• pp.59-63
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• 1999

A cDNA encoding rice chloroplast small HSP, Oshsp21, was introduced into Escherichia coli using the pET expression vector to analyze the possible function of Oshsp21 under heat stress. We compared the viability of E. coli ${\lambda}BL21$ (DE3) cells transformed with recombinant plasmid containing Oshsp21 with the control E. coli cells transformed with pET28a vector under heat stress after IPTG induction. Upon heat treatment at $50^{\circ}C$, those cells that expressed Oshsp21 showed improved viability compared with control cells. When the cell lysates from E. coli transformants were heated at $55^{\circ}C$, the amounts of proteins denatured in the control and pEhsp21-transformed cells were about 60% and 35% of total cell proteins, respectively. These results indicate that rice chloroplast small HSP function as a molecular chaperone in cells.

• Korean journal of applied entomology
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• v.35 no.4
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• pp.321-325
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• 1996

To isolate a novel gene for antibacterial peptide, an inducible clone(BmInc8) was selected by differential screening strategy from Bombyx mori cDNA library prepared from lavae injected with Escherichia coli. This clone contained a cDNA insert of 564 nucleotides and encoded 59 amino acids with an apparent molecular mass of 6.3 kDa. The cDNA sequence of BmInc8 had 61.2% identity compared to that of the bactericidin from Manduca sexta and also the deduced amino acids sequences from this insert had 65% identity compared to that of the cecropin D peptide Hyalophora cecropia. The transient expression assay of this insert using prokaryotic expression vector system revealed that the expressed peptide displayed the antibacterial activity. The cDNA sequence was deposited in GenBank under the accession number U30289.