• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6709-6714
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• 2013

Background: Breast cancer is a leading cause of death in women. The available chemotherapy drugs have been associated with many side effects. Bromelain has novel medicinal qualities including anti-inflammatory, anti-thrombotic, fibrinolytic and anti-cancer functions. Commercially available bromelain is obtained through tedious methods; therefore, recombinant bromelain may provide a cheaper and simpler choice with similar quality. Materials and Methods: This study aimed to assess the effects of commercial and recombinant bromelain on the cytokinetic behavior of MCF-7 breast cancer cells and their potential as therapeutic alternatives in cancer treatment. Cytotoxic activities of commercial and recombinant bromelain were determined using (sulforhodamine) SRB assay. Next, cell viability assays were conducted to determine effects of commercial and recombinant bromelain on MCF-7 cell cytokinetic behavior. Finally, the established growth kinetic data were used to modify a model that predicts the effects of commercial and recombinant bromelain on MCF-7 cells. Results: Commercial and recombinant bromelain exerted strong effects towards decreasing the cell viability of MCF-7 cells with $IC_{50}$ values of 5.13 ${\mu}g/mL$ and 6.25 ${\mu}g/mL$, respectively, compared to taxol with an $IC_{50}$ value of 0.063 ${\mu}g/mL$. The present results indicate that commercial and recombinant bromelain both have anti-proliferative activity, reduced the number of cell generations from 3.92 to 2.81 for commercial bromelain and to 2.86 for recombinant bromelain, while with taxol reduction was to 3.12. Microscopic observation of bromelain-treated MCF-7 cells demonstrated detachment. Inhibition activity was verified with growth rates decreased dynamically from 0.009 $h^{-1}$ to 0.0059 $h^{-1}$ for commercial bromelain and to 0.0063 $h^{-1}$ for recombinant bromelain. Conclusions: Commercial and recombinant bromelain both affect cytokinetics of MCF-7 cells by decreasing cell viability, demonstrating similar strength to taxol.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.174-182
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• 1992

The hemagglutinin-esterase glycoprotein (HE) gene of bovine coronavirus, coupled with a simian virus 40 early promoter and polyadenylation signal, was inserted into a human adenovirus transfer vector. The transfer vector was used to co-transfect 293 cells along with adenovirus genomic DNA. The hemagglutinin-esterase transcription unit was rescued into the adenovirus genome by homologous in vivo DNA recombination between the vector plasmid DNA and the adenovirus genomic DNA, and a recombinant adenovirus was isolated by several rounds of plaque assays. Thus the recombinant adenovirus carries the hemagglutinin-esterase gene in the early transcription region 3 (E3) of the adenovirus genome in the parallel orientation to the E3 transcription. The recombinant adenovirus synthesized the HE polypeptide in HeLa cells as demonstrated by immunoprecipitation with anti-coronavirus rabbit antisera. The recombinant HE polypeptide could be labelled by $[^3H]$glucosamine, demonstrating that the recombinant HE was glycosylated. Cells expressing the HE polypeptide exhibited hemadsorption activity when incubated with mouse erythrocytes. The HE was transported to the plasma membrane as shown by the cell surface immunofluorescence, indicating that the recombinant HE polypeptide retained its biological activities. Potential for the use of infectious recombinant adenovirus as a live virus-vectored vaccine candidate for bovine coronavirus disease is discussed.

• KSBB Journal
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• v.9 no.2
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• pp.165-173
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• 1994

In this paper we have described the structural characterization of recombinant bovine somatotropin produced in Escherichia coli. Recombinant bovine somatotropin consists of 191 amino acid residues with a calculated molecular weight of 21,802 Da. For fragmentation of recombinant bovine somatotropin, we have used trypsin, Staphylococcus aureus V8 pretease, CNBr, and mild acid hydrolysis method. Digestion and cleavage with these proteases and chemicals yielded peptides of various size for amino acid sequence determination. The N-terminal sequence analysis was carried out up to thirty residues. Because the design of the recombinant bovine somatotropin gene for expression was such that the coding sequence begins with an initiation codon, AUG, before Ala, the first amino acid of bovine somatotropin, we could expect the initial amino acid as N-formyl Met. But the first amino acid of this protein, expressed in E. coli cells as inclusion bodies, was Ala. And the amino acid composition of RP-HPLC purified recombinant bovine somatotropin was determined and no essencial difference was observed. The amino acid sequence of the recombinant bovine somatotropin was identical to that predicted from its recombinant gene. There was no processing or replacement of amino acid residues in recombinant bovine somatotropin expressed in E. coli. The hydropathy plot of recombinant bovine somatotropin revealed a hydrophobic region at the NH2-terminus and hydrophilic region at the COOH-terminus. The E. coli expression system is thought to be valuable for the expression of recombinant bovine somatotropin because protein was processed to remove the N-terminal Met residue by methionyl-aminopeptidase autonomously.

• Journal of Plant Biotechnology
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• v.4 no.3
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• pp.95-101
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• 2002

Molecular faming has the potential to provide large amounts of recombinant protein for use in diagnostics and as therapeutics. Various strategies have been developed to enhance the expression level, stability, and native folding of recombinant proteins produced in plants. Few investigations into the subcellular distribution of recombinant proteins within plant cells have been published despite the potential to increase the expression level and impact the purification process. This review article discusses the current strategies used for targeting recombinant proteins to various subcellular locations and the advantages of targeting to seed oil bodies for molecular farming applications. Specifically, the affinity capture of antibodies using recombinant oilbodies is discussed.

• Journal of fish pathology
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• v.33 no.2
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• pp.163-169
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• 2020

Snakehead rhabdovirus (SHRV) is different from other fish novirhabdoviruses such as viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV) in that it replicates at high temperatures. Therefore, the delivery of foreign proteins to fish living at high water temperature would be possible by using recombinant SHRVs. In the present study, to evaluate the possible use of SHRV as a vehicle for foreign proteins delivery, we generated a recombinant SHRV that contains an enhanced-GFP (eGFP) gene between nucleoprotein (N) and phosphoprotein (P) genes (rSHRV-A-eGFP), and another recombinant SHRV expressing two heterologous genes by inserting an eGFP gene between N and P genes, and mCherry gene between P and M genes (rSHRV-AeGFP-BmCherry). Epithelioma papulosum cyprini (EPC) cells infected with the recombinant SHRVs showed strong fluorescence(s), suggesting the possible availability of recombinant SHRVs for the development of combined vaccines by expressing multiple foreign antigens.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2013.05a
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• pp.1006-1008
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• 2013

A novel recombinant baculovirus vector system containing coding genes for polyhedron promoter, vesicular stomatitis virus G (VSVG), polyA, cytomegalovirus (CMV) promoter, enhanced green fluorescent protein (EGFP), and protein transduction domain (PTD) was constructed. We applied this recombinant baculovirus vector into cells and murine tissues and compared efficacy of gene transfer and expression of this recombinant baculovirus vector system with control vector system. From this result, we confirmed that this novel recombinant baculovirus vector system was very effective than control vector system.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.738-744
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• 2003

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.685-689
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• 2009

We describe the expression of recombinant canstatin from stably transformed Bombyx mori BmS (BmS) cells. Recombinant canstatin was secreted into a culture medium with a molecular mass of approximately 29 kDa. Densitometric scanning showed that the secreted canstatin accounted for approximately 91% of the total canstatin production. Recombinant canstatin was also purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. The identity of the purified protein was confirmed as human canstatin by nano-LC-MS/MS analysis. Purified recombinant canstatin inhibited human endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition ($ED_{50}$) for recombinant canstatin expressed in stably transformed BmS cells was approximately 0.64 ${\mu}g/ml$. A maximum production level of 11 mg/l recombinant canstatin was obtained in a T-flask culture of BmS cells after 6 days of incubation.

• Korean Journal of Microbiology
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• v.25 no.1
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• pp.46-51
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• 1987

In order to increase the production of tryptophan by maximizing expression of recombinant trp plasmid, Klebsiella pneumoniae KC 105(pheA tyrA trpE trpR tyrR) was genetically modified. KC 107, inosine monophospate(IMP) auxotroph from KC 105 and KC 108, histidine(His) auxotroph from KC 107 were also derived respectively to increase phosphoribosylpyrophosphate(PRPP) production which is required for tryptophan biosynthesis. From KC 107 phosphoribosylpyrophosphate consumption which is required for tryptophan biosynthesis. From KC 107 and KC 108, KC 109 and KC 110, both arginine auxotrophs were derived respectively. To investigate the expression of recombinant trp plasmid in the selected K. pneumoniae mutants, the auxotrophic mutants were transformed with recombinant trp plasmids pSC 101-$trpE^{FBR}$, pSC 101-trpL(.DELTA.att) $trpE^{FBR}$ (pSC 101-trp-AF). Amount of tryptophan produced and activities of tryptophan synthase of $trpR^{-}$ mutant (KC 100) and $tyrR^{-}$ mutnat(KC 105) containing recombinant plasmid pSC 101-trp operon were increased by 30-40% as compared with KC 99(pheA tyrA trpE) containing recombinant plasmid pSC 101-trp operon. Activities of tryptophan synthase and production of tryptophan of KC 108 ($His^{-}$) and KC 109($Arg^{-}$) containing recombinant plasmid pSC 101-trp operon were increase by two-fold as compared with KC 107 containing pSC 101-trp operon.

• The Journal of Korean Society of Virology
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• v.29 no.3
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• pp.145-153
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• 1999

We have now constructed a novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) producing polyhedra with Bacillus thuringiensis (Bt) CryIAc crystal protein. The recombinant polyhedra produced by the recombinant baculovirus, Btrus, in insect cells was characterized. The recombinant baculovirus has two independent transcription units in opposite orientations with two promoters, p10 or polyhedrin gene promoter each initiating transcription of either native polyhedrin or fusion protein with polyhedrin and Bt Cry1Ac crystal protein. Surprisingly, this recombinant baculovirus stably produced recombinant polyhedra which were nearly similar to those of wild-type AcNPV. The immunogold staining experiment showed that the recombinant polyhedra were assembled with polyhedrin and Bt Cry1Ac crystal protein, and contained virus particles. Insecticidal toxicity of recombinant polyhedra of Btrus to the fall webworm, Hyphantria cunea, was strikingly improved in comparison with the wild-type AcNPV.