• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.1931-1937
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• 2019

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.

• Journal of Life Science
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• 제12권2호
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• pp.53-56
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• 2002

The baculovirus expression vector system (BEVS) is one of the powerful heterologous protein expression systems using insect cells. As a result this has become a hot issue in the fleld of biotechnology. The advantage of the BEVS is that the large-scale production of heterologous proteins, which undergo posttranslational modification in the endoplasmic reticulum (ER), can be accomplished. Altrough posttranslational modification of heterologous proteins in insect cells is more similar to mammalian cells than yeast, it is not always identical. Therefore, aggregation and degradation can sometimes occur in the ER. To produce a high level of bioactive heterologous proteins using BEVS in insect cells, the prerequisite is to completely understand the posttranslational conditions that determine how newly synthesized polypeptides are folded and assembling with ER chaperones in the ER lumen. Here, we provide information on current BEVS problems and the possibility of successful heterologous protein production from mammalian cells.

• Asian-Australasian Journal of Animal Sciences
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• 제13권7호
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• pp.894-896
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• 2000

In vitro fertilization can be used for salvaging superior buffalo germplasm which otherwise goes waste after the slaughter of animals. This technology has also increased our basic understanding of growth of germ cells and embryos. The requirement of growing embryos is peculiar and stage specific. In the present study the cleavage and development of buffalo embryos were studied with homologous (buffalo) and heterologous (goat) oviductal cell co-culture systems. The cleavage rate improved significantly (p<0.01) in both homologous and heterologous co-culture as compared to control (55.3, 46.8 and 11.4%). The morula formation using homologous and heterologous oviductal cells also increased significantly as compared to control group (43.6, 21.9 & 1.9%). There was no blastula formation in control group, but addition of oviductal cells either from homologous or heterologous species significantly increased the blastula formation (9.5, 12.5%). The cleavage rate and embryo development was slightly better (non significant) in homologous as compared to heterologous oviductal cell culture. It was concluded that the use of oviductal cell co-culture (homologous and heterologous species) have significantly improved cleavage and development of buffalo embryos in vitro.

• KSBB Journal
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• 제16권1호
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• pp.15-23
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• 2001

The development of expression systems for heterologous proteins has been greatly demanded not only for the study of the structure/function relationships of these proteins but also for their biotechnological and pharmaceutical applications. During the past decades, the methylotrophic yeast Hansenula polymorpha and Pichia pastoris have drawn attention as one of promising hosts for the production of a variety of heterologous proteins. The increasing popularity of H. polymorpha and P. pastoris as the host systems can be attributed to the several advantages over the traditional yeast Saccharomyces cerevisiae, such as the availability of very strong and tightly regulated promoters from the enzymes involved in the metabolism of methanol, a very high-cell density even on simple mineral media, and a high stability of expression plasmids. Furthermore, it has been observed that glycoproteins from these two yeasts are less hyperglycoylated compared to those from S. cerevisiae. Despite substantial similarities as methylotrophic yeasts, however, these two expression systems have some unique features distinguished from each other. In this paper we present a brief overview on the present status of the expression systems developed in methylotrophic yeast, mainly focusing on the similarities and differences between the H. polymorpha and P. pastoris systems.

• Genomics & Informatics
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• 제1권2호
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• pp.113-118
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• 2003

Among a number of antigens characterized in M leprae, an etiological agent of Leprosy, the 18 kDa antigen, is unique to M leprae. We have previously determined a sequence specific element in the 18 kDa gene of M leprae, which confers transcriptional repression. In this report, we have examined if the element could be applied to genes other than the 18 kDa gene of M leprae. To identify the roles of the regulatory sequence in heterologous promoter, we have constructed pB3 vector series, which contains BCG hsp65 promoter and the M leprae 18 kDa transcription repression responsive element in tandem using LacZ gene as a reporter gene. Cloning of hsp65 promoters of M bovis BCG or M smegmatis in front of LacZ gene resulted in normal $\beta$­galactosidase activity as expected. However, when the sequence element was placed between the promoter and the LacZ gene, $\beta$-galactosidase activity was reduced 10-fold less. Also we have examined with pB3(-) vector, that harbors the transcription repression responsive element in a reversed orientation, the $\beta$-galactosidase activity was found to be similar to pB3(+) vector. Thus, these results further confirm that M leprae 18 kDa transcription repression responsive element could regulate BCG hsp65 heterologous promoter and that the element could act as an operator for the transcription of mycobacteria.

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.161-167
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• 2010

Early diagnoses of pregnancy for animal such as swine and bovine is extremely important to increase income of a farmhouse and for the management of farm. For the development of immunoasaay system of pregnancy in swine, we report a competitive heterologous enzyme linked immunosorbent assay (ELISA) for the direct measurement of oestrone sulfate (E1S) in diluted urine using anti-E1G (glucuronide) monoclonal antibody which cross react with ElS. The principle of assay was based on the typical solid-phase competitive ELISA methods using E1G-HRP (horseradish peroxidase) as a tracer and E1S for standard. The method had a reasonable sensitivity for the detection of E1S with 0.15 ng/ml as a detection limit. The intra-assay and inter-assay precisions were raging coefficient of from 8.50~9.67% and 8.50~9.87%, respectively, which were quite acceptable. In a field trial with a group 37 sows (18 non-pregnancy and 19 pregnancy sows) after day 29~30 post service, the concentration of E1S were determined to be below 30 ng/ml in all non-pregnancy group and over 48 ng/ml in pregnancy group except one sample. The method described here, heterologous ELISA for the measurement of E1S in urine is good enough for monitoring the early pregnancy test of swine.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.988-998
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• 2015

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl₂-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30^oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

• Journal of Microbiology and Biotechnology
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• 제3권2호
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• pp.65-72
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• 1993

A heterologous gene expression and secretion system using a methylotrophic yeast, Hansenula polymorpha was developed for the production of anticoagulant hirudin. Hirudin gene was expressed under the control of a strong and inducible methanol oxidase (MOX or AOX) promoter. The mating factor a pre-pro leader sequence of Saccharomyces cerevisiae was employed for hirudin to be secreted into the extracellular medium. Hirudin expression cassette was introduced into three strains of H. polymorpha, A16, HPBl and DLl which have different genetic backgrounds. This expression cassette was stably integrated into the host chromosomal DNA. Biologically active and mature hirudin was efficiently expressed and secreted into the extracellular medium. About 19 mg/L of hirudin was found in the culture supernatant in the case of a two-copy integrant of the strain HPBl under suboptimal culture conditions.

• 한국미생물·생명공학회지
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• 제17권1호
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• pp.81-83
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• 1989

The flagella antigenic variation of nine Bacillus thuringiensis serovar kurstaki temperature-sensitive mutants grown at the permissive temperature (3$0^{\circ}C$) was detected by a serological agglutination between H-antigen and antiserum. The flagella antigens were injected to rabbits to prepared their antisera, and then their homologous and heterologous titers of the antisera were measured. The homologous titers were ranged from 1:6,400 to 1:12,800, but the heterologous titers were very low. The H-antigen of the wild type strain was not agglutinated to 4 heterologous antisera, ts-U23 not to 7, ts-U3l not 5, ts-U32 not to 4, ts-U33 not to 7, ts-U7l not to 4, ts-U73 not to 6, ts-U74 not to 6, ts-U91 not to 4 and ts-U603 not to 4 antisera.

• 대한수의학회지
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• 제61권2호
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• pp.11.1-11.5
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• 2021

To evaluate avian hepatitis E virus (aHEV) as an RNA vaccine platform, ORF2 of aHEV was replaced by heterologous genes, such as eGFP and HA-tag, in aHEV infectious cDNA clones. eGFP and HA-tag replicons were expressed in LMH cells. To confirm expression of the heterologous protein, ORF2 was replaced with the antigenic S1 gene of IBV. The IBVS1 replicon was expressed in LMH cells. To our knowledge, this is the first investigation showing the potential as a RNA vaccine platform using an aHEV. In the future, it may be used in the development of RNA vaccines against various pathogens.