• KSBB Journal
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• v.5 no.3
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• pp.293-298
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• 1990

Alpha-interferon was produced by using recombinant Escherichia coli strains, which carry cloned alpha-interferon gene in plasmid vectors, pIF-III-B and pIF-III-C. With the aid of signal sequence of E. coli lipoprotein, which is placed right in front of the upstream of the cloned alpha-interferon gene of the plasmids, about 50% of alpha-interferon produced was excreted or secreted. Meanwhile, there was no extracellular production of alpha-interferon from the recombinant strain carrying the plasmid Hif-2h that lacks the signal sequence of lipoprotein.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1898-1903
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• 2007

We attempted to modulate the overall protein expression rate through the addition of a repressor against the araBAD promoter system of Escherichia coli, in which glucose was used as a repressor. Therefore, 0.5% L-arabinose was initially contained as an inducer in culture medium, and either 2% glucose or 2% glycerol was used as a carbon source, and it was found that the expression of recombinant interferon-${\alpha}$ could be observed at the beginning of the batch culture when glycerol was used as a carbon source. However, when glucose was used, the initiation of recombinant interferon-${\alpha}$ expression was delayed compared with that when glycerol was used. Furthermore, when the addition of 0.5% glucose was carried out once or twice after 0.5% L-arabinose induction during DO-stat fed-batch culture, the distributions of soluble and insoluble recombinant interferon-${\alpha}$ were modulated. When glucose was not added after the induction of L-arabinose, all of the expressed recombinant interferon-${\alpha}$ formed an inclusion body during the later half of culturing. However, when glucose was added after induction, the expressed recombinant interferon-${\alpha}$ did not all form an inclusion body, and about half of the total recombinant interferon-${\alpha}$ was expressed in a soluble form. It was deduced that the addition of glucose after the induction of L-arabinose might lower the cAMP level, and thus, CAP (catabolite activator protein) might not be activated. The transcription rate of recombinant interferon-${\alpha}$ in the araBAD promoter system might be delayed by the partial repression. This inhibition of the transcription rate probably resulted in more soluble interferon-${\alpha}$ expression caused by the reduction of the protein synthesis rate.

• KSBB Journal
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• v.6 no.1
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• pp.9-14
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• 1991

We studied the production and excretion of alpha-interferon in recombinant Escherichia coli harboring plasmid pIF-III-B, which carries alpha-interferon gene under the control of lipoprotein and lacUV5 promoter, and lac operator. Basically, the effects of concentrations of ampicillin and an inducer, IPTG, for the expression of the cloned gene, on the productions of alpha-interferon and plasmid stability were studied. The highest production of alpha-interferon was observed at 50 mg/1 of ampicillin concentration and 0.5 mM of IPTG. The plasmid pIF-III-B was maintained very stably in medium with ampicillin but segregated rapidly in medium without ampicillin. Also, the plasmid was segregated more rapidly in medium with an inducer higher than 0.5 mM.

• Journal of Microbiology and Biotechnology
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• v.12 no.6
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• pp.916-920
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• 2002

This report describes a high-level expression of human alpha-2a interferon ($IFN{\alpha}-2a$) in Escherichia coli and its pilot scale purification by using a monoclonal antibody-independent chromatographic procedure that is based on anion-exchange, cation-exchange, hydrophobic interaction, and gel filtration. The recombinant E. coli produced much more $IFN{\alpha}-2a$ in a soluble form, when cultivated at low temperatures than at high-temperature fermentation. However, if the bacterial growth was taken into consideration, fermentation at $30^{\circ}C$ seemed optimal for the interferon production. By using our new protocol, we recovered approximately 160 mg of $IFN{\alpha}-2a$ with a specific activity of $3.59{\times}10^8$ IU/mg from 201 of the broth. The gel permeation chromatographic and SDS-PAGE indicated that the interferon preparation was purified to homogeneity and was of the correctly folded fast-migrating monomer.

• KSBB Journal
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• v.5 no.3
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• pp.195-200
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• 1990

The growth behavior of recombinant Escherichia coli cells having plasmid pIF-III-B, which carries human alpha-interferon gene under the control of lpp promoter, lac promoter and lac operator, was studied by using of various E. coli host strains. Expression of the alpha-IFN gene is controllable by using inducer IPTG because the plasmid also contains lacI gene which produces lac regressors. The repressors block the transcription of alpha-IFN gene. There were considerable differences in cell growth according to the host strains used. Cell growth was inhibited not only by plasmid pIF-III-B itself but also by the induction of alph-a-IFN gene expression. Growth inhibition caused by the plasmid itself was more serious than that caused by the induction of alpha-IFN gene expression.

• BMB Reports
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• v.28 no.6
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• pp.477-483
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• 1995

The recombinant human interferon alpha 2a ($rhIFN-{\alpha}2a$), expressed in Saccharomyces cerevtsiae, was purified from insoluble aggregates. The inclusion body of $rhIFN-{\alpha}$ was solubilized by guanidine salt in the presence of disulfide reducing agent. The refolding of denatured $rhIFN-{\alpha}2a$ was achieved by simple dilution. The authentic interferon alpha, which has two correctly matched disulfide bonds, was seperated from incompletely oxidized $IFN-{\alpha}$ and dimeric $IFN-{\alpha}$ by use of a CM-Sepharose column, followed by size exclusion columns at two different pH conditions. The purified protein has been subjected to detailed physicochemical characterization including sequence determination. Unlike other $rhIFN-{\alpha}2a$ from E. coli reported, the $rhIFN-{\alpha}2a$ from S. cerevisiae has no methionine residue at its N-terminus originating from the start codon, ATG. The pI of the protein was determined to be 6.05 with a single band in the pI gel, which demonstrated that the purified $rhIFN-{\alpha}$ was homogeneous. The structural study using circular dichroism showed that the protein retains its three dimensional structure in the wide range of pH conditions between pH 3 and 9, and only minor strucural deformation was observed at pH 1.0.

• Biomolecules & Therapeutics
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• v.1 no.2
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• pp.266-269
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• 1993

The acute toxicity of a recombinant human interferon $\alpha$A (code name: LBD-007) was evaluated in both sexes of ICR mice, 5 weeks old, by the oral, subcutaneous and intravenous routes of administration. Based on the results, LBD-007 was not considered to induce any toxic effect on the mice in mortalities, clinical findigs, body weights and gross findings. It is suggested that LD$_{50}$ values in mice would be $>48{\times}10^8$ IU/kg in the oral, subcutaneous or intravenous routes.s.

• Toxicological Research
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• v.9 no.1
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• pp.119-123
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• 1993

LBD-007, a newly developed recombinant human interferon ${\alpha}$A, was tested for primary eye and skin irritation in male New Zealand White rabbits. In the primary eye irritation test, 0.1ml of a solution of LBD-007 was instilled into the eye. In rinsing group, the eye was washed with water 30 seconds after instillation. No reaction was observed at the cornea, iris and conjunctivae by LBD-007. In the primary skin irritation test, LBD-007 was applied to the back of rabbits for 24 hours. Primary irritation index was "0" in test and control sites of all animals. Thus LBD-007 was evaluated as a non-irritant on the basis of the criteria of Draize et al.,(1994).

• YAKHAK HOEJI
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• v.53 no.3
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• pp.101-106
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• 2009

Recombinant interferon alpha-2a is an active formula for the treatment of various cancer cells like malignant melanoma and a variety of virus infection diseases like acute or chronic hepatitis. The bioassay system based on the measurement of virus inhibitory activity by interferon has been used for interferon analysis with low repeatability. Here, we developed the HPLC assay to measure reproducibly interferon alpha-2a protein content which is replaceable to the reported bioassay. This method separated interferon alpha-2a from its oxidized forms and human serum albumin used as excipients. The regression coefficient using interferon alpha-2a EP CRS is more than 0.9 in the range from 5 ${\mu}g/ml$ to 200 ${\mu}g/ml$. The recovery result in the range from 15 ${\mu}g/ml$ to 60 ${\mu}g/ml$ is $97{\sim}104%$ and the precision is $0.2{\sim}1.7%$. The interferon alpha contents of 5 products are about 30 ${\mu}g/ml$.

• BMB Reports
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• v.29 no.4
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• pp.365-371
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• 1996

Partially reduced interferon-a ($IFN-{\alpha}$) was cross-linked with thymosin ${\alpha}1$ ($T{\alpha}1$) using sulfo-succinimidyl (4-iodoacetyl) amino benzoate (SIAB), a bifunctional cross-linking reagent. The partially reduced $IFN-{\alpha}$ optimal for the cross-linking reaction was obtained by incubating native $IFN-{\alpha}$ with 0.5 mM DTT at $30^{\circ}C$ for 60~100 min. $T{\alpha}1$ was activated by incubating with sulfo-SIAB at $37^{\circ}C$ for 30 min to produce $T{\alpha}1-IAB$. The $T{\alpha}1-IFN-{\alpha}$ cross-linking was achieved by the reaction of the partially reduced $IFN-{\alpha}$ with $T{\alpha}1-IAB$. This cross-linking was between the sulfhydryl group of Cys1 in $IFN-{\alpha}$ and the N-terminal amino group of $T{\alpha}1$ through acetyl amino benzoate as a spacer. The immunological activity of the cross-linked molecule showed the same extent as that of $T{\alpha}1$, and most of the antiviral activity was retained compared to that of the partially reduced $IFN-{\alpha}$.