• Biotechnology and Bioprocess Engineering:BBE
• /
• 제2권2호
• /
• pp.86-89
• /
• 1997

Epidermal growth factor(EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector in which the gene product was supposed to be expressed with 6$\times$His tag for the subsequent purification. The recombinant mature EGF was expressed in M15[Rep4], an Escherichia coli host strain, in amount of 30-40% of total proteins pressent in E. coli extract by the addition of isopropylthio-$\beta$-galactopyranoside (IPTG). The recombinant EGF purified using a Ni2+-NTA affinity colume chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate proteins when murine NH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology and information for the production of recombinant EGF.

• Molecules and Cells
• /
• 제46권9호
• /
• pp.558-572
• /
• 2023

Chronic obstructive pulmonary disease (COPD) will be the third leading cause of death worldwide by 2030. One of its components, emphysema, has been defined as a lung disease that irreversibly damages the lungs' alveoli. Treatment is currently unavailable for emphysema symptoms and complete cure of the disease. Hyaluronan (HA) and proteoglycan link protein 1 (HAPLN1), an HA-binding protein linking HA in the extracellular matrix to stabilize the proteoglycan structure, forms a bulky hydrogel-like aggregate. Studies on the biological role of the full-length HAPLN1, a simple structure-stabilizing protein, are limited. Here, we demonstrated for the first time that treating human alveolar epithelial type 2 cells with recombinant human HAPLN1 (rhHAPLN1) increased TGF-β receptor 1 (TGF-β RI) protein levels, but not TGF-β RII, in a CD44-dependent manner with concurrent enhancement of the phosphorylated Smad3 (p-Smad3), but not p-Smad2, upon TGF-β1 stimulation. Furthermore, rhHAPLN1 significantly increased sirtuins levels (i.e., SIRT1/2/6) without TGF-β1 and inhibited acetylated p300 levels that were increased by TGF-β1. rhHAPLN1 is crucial in regulating cellular senescence, including p53, p21, and p16, and inflammation markers such as p-NF-κB and Nrf2. Both senile emphysema mouse model induced via intraperitoneal rhHAPLN1 injections and porcine pancreatic elastase (PPE)-induced COPD mouse model generated via rhHAPLN1-containing aerosols inhalations showed a significantly potent efficacy in reducing alveolar spaces enlargement. Preclinical trials are underway to investigate the effects of inhaled rhHAPLN1-containing aerosols on several COPD animal models.

• BMB Reports
• /
• 제29권3호
• /
• pp.266-271
• /
• 1996

A recombinant human erythropoietin (EPO), expressed in Chinese hamster ovary (CHO) cells, is glycosylated at Asn 24, Asn 38, Asn 83, and Ser 126. After release of the N-linked carbohydrate chains by $peptide-N^{4}-(N-acetyl-{\beta}-glucosaminyl)$ asparagine amidase F, the oligosaccharides were analyzed by FACE (Fluorophore-Assisted Carbohydrate Electrophoresis). The O-linked carbohydrate chain was separated by hydrazine, and analyzed by FACE. The monosacccharide composition of recombinant EPO showed man nose, fucose, galactose, N-acetylglucosamine, N-acetylneuraminic acid, and a trace of N-acetylgalactosamine, which are typical monosaccharides in the glycoproteins from the CHO cell. Sequences of N-linked and O-linked oligosaccharides were determined. The structure and composition of oligosaccharides attached to recombinant human EPO, expressed in the CHO cell, are identical to the reported oligosaccharide structure in human EPO isolated from urine.

• 대한수의학회지
• /
• 제37권3호
• /
• pp.547-554
• /
• 1997

Galectin-3 is known as an animal ${\beta}$-galactoside-binding lectin charicterized with S-type carbohydrate recognition domain. It plays a role in growth, adherence and movement of cells. It is, also, related to the cell transformation and metastasis of tumor cells. In this study, we have expressed and purified recombinant human galectin-3 (rHgalectin-3) using E coli system and asialofetuin affinity chromatography for the future development of monoclonal antibody to Hgalectin-3, which is suggested as the tumor marker for the gastric and thyroid gland cancers. Expressed protein was confirmed as the Hgalectin-3 by immunoblot with cross-reactive murine monoclonal antibody. Lectin activity and specificity of purified protein were, also, confirmed by the competitive inhibition with galectin-3 specific carbohydrate, lactose. Like physiological galectin-3, lectin activity of the molecule was not changed in nonreduced condition. Dimer formation, furthermore, was observed at high concentration of the protein even in the reduced condition, which is well known in physiological galectin-3. These results showed purified rHgalectin-3 has the same activity and molecular nature compared to the physiological galectin-3.

• Journal of Microbiology and Biotechnology
• /
• 제2권2호
• /
• pp.135-140
• /
• 1992

The partitioning of recombinant human interleukin-2(rhII-2) in PEG 8000-dextran 38800 aqueous two-phase system has been investigated using three different sources of rhIL-2. In the case of pure rhIL-2, the solubility in a PEG-dextran two-phase system was low and most of rhIL-2 was partitioned into the bottom phase. For the recovery of rhIL-2 from insoluble protein aggregates, the inclusion bodies of recombinant E. coli were solubilized by the treatment with sodium dodecyl sulfate (SDS). The addition of SDS significantly enhanced not only the solubility of rhIL-2 but also the partitioning of rhIL-2 to the top phase. When the ratio of SDS to rhIL-2 was 2.0, the partition coefficient(K) and the recovery yield(Y) at the top phase were 4.5 and 88%, respectively, at pH 6.8. In order to reduce the recovery steps further, SDS was directly added to the intact recombinant E. coli cells and then partitioned into the PEG/dextran aqueous two-phase system. The observed partition coefficient ($K{\cong{3.0$) and recovery yield ($Y{\geq}80%$ )of this method were comparable to the rhIL-2 recovery from insoluble protein aggregates. The results obtained in this work indicate that PEG-dextran two-phase partitioning might provide a simple way for the recovery and partial purification of recombinant proteins which are produced as inclusion bodies.

• IMMUNE NETWORK
• /
• 제16권5호
• /
• pp.296-304
• /
• 2016

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation.

• Animal cells and systems
• /
• 제1권2호
• /
• pp.317-321
• /
• 1997

Tau protein is one of the microtubule-associated proteins in the mammalian brain. In Alzheimer's disease, tau protein is immobilized in the somatodendritic compartment of certain nerve cells, where it forms a part of the paired helical filament (PHF). To understand the role of tau protein in the formation of PHF, a recombinant human tau protein expressed in Escherichia coli and five synthetic peptide fragments (peptide 1 to peptide 5), corresponding to the C-terminal region of tau protein, were prepared and their ability in self-assembly to form filamentous structures was examined. The recombinant human tau protein formed short rod-like structures in 0.1M MES buffer containing 1 mM $MgCI_2$, while a synthetic peptide fragment 1 containing 55 amino acid residues could assemble into a lot of long filamentous structures in water and particularly twisted helical structures in 0.1M MES buffer containing 1 mM $MgCI_2$. This suggests that the C-terminal region possesses a filament-forming ability and may be related to the formation of the helical structure by providing a powerful filament-forming driving force.

• 한국식품영양과학회지
• /
• 제28권2호
• /
• pp.348-352
• /
• 1999

We have intended to accelerate the secretion of human lysozyme(HLY) with permeabilizing agents from the cultivated cells of the recombinant Saccharomyces cerevisiae. The five agents CaCl2, Tween 80, ethanol, Triton X 100, and cetyltrimethylammonium bromide(CTAB) were used as permeabilizing agents. Treatments of the yeast cell with CaCl2, Tween 80, and ethanol were effective to increase the secretion from the yeast cells. Especially, treatment of 10% ethanol increased the extracellular HLY activity by 38.6% at 30oC for 48 h in culture broth. But Triton X 100 and CTAB unexpectedly didn't play a role in increase of HLY secretion. Recovery of a foreign protein by permeabilizing agents is easier than by osmotic shock, and is less expensive than enzymatic digestion.

• Journal of Yeungnam Medical Science
• /
• 제24권2호
• /
• pp.262-274
• /
• 2007

AdBMP-2와 AdBMP-7을 형질도입 시킨 사람 섬유아세포와 사람 골수기질세포를 면역결핍 생쥐의 척추 옆 근육으로 주입하여 척추 유합을 유도한 결과, AdBMP-7/BMSC가 AdBMP-2/BMSC 또는 AdBMP-7/HuFb와 AdBMP-2/HuFb 보다 골 형성능이 우수하였으며, 척추 유합을 잘 유도하였음을 확인하였다.

• Journal of Microbiology and Biotechnology
• /
• 제11권2호
• /
• pp.234-241
• /
• 2001

The C-terminal 393 bp region of the human interleukin-16 (IL-16) gene was cloned and expressed in E. coli along with mammalian cell lines. Recombinant IL-16 expressed from E. coli was 22 kDa on SDS-PAGE and showed 260% of chemoattractant activity at a concentration of $0.1\;{\mu}g/ml$. HeLa, COS, and Neuro-2a cells were transduced by recombinant retrovirus vector pLNC/IL-16/IRES/TK and the intracellular and secreted amounts of IL-16 produced by HeLa/IL-16/TK, COS/IL-16/TK, and Neuro-2a/IL-16/TK cells were determined by enzyme-linked immunosorbent assay (ELISA). HeLa/IL-16/TK $(1{\times}10^5)$ and COS/IL-16/TK $(1{\times}10^5)$ cells secreted 36.1 and 13.3 ng of IL-16 for 48 h, respectively. Forty-nine ng and 86.4 ng of IL-16 remained in the cell lysates of HeLa/IL-16/TK and COS/IL-16/TK. Intracellular and secreted amounts of IL-16 from Neuro-2a/IL-16/TK $(5{\times}10^5)$ cells during 24 h cultivation were 50 ng and 3.3 ng, respectively. Also, HeLa and COS cells wee stably transfected with mammalian expression vector pCRIII/IL-16. Both culture media and cell lysates prepared from HeLa/IL-16 cells and COS/IL-16 cells showed chemoattractant activity ranging from 190% to 460% as compared to the control experiment. Expression of the herpes simplex virus thymidine kinase (HSV0tk) gene in pLNC/IL-16/ IRES/TK bicistronic retroviral expression vector was verified by performing a genciclovir (GCV) sensitivity assay. Finally, IL-16 repressed Tat-transactivated human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) promoter activity.