• Membrane Journal
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• v.19 no.2
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• pp.113-121
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• 2009

Porous affinity chitosan and chitin membranes with good mechanical strength and high protein binding capacity were prepared by using silica particles as porogen. The maximum binding capacity of affinity chitosan membrane for BSA protein is 21.8mg/mL, and that of affinity chitin membrane for lysozyme enzyme is 26.1mg/mL. Chromatographic separations of BSA and lysozyme proteins using the porous affinity chitosan and chitin membranes were performed with change of the flow rate, loading amount and concentration of protein loading solutions. Protein eluted amount and binding yield were calculated from the filtration chromatograms consisted of loading/washing/elution sequences. Protein binding amount and yield were increased with decreasing of flow rate, increasing of loading amount and concentration of protein loading solutions. Those results suggest that the porous chitosan and chitin membranes prepared by using silica particles as porogen are suitable in affinity filtration chromatography for large scale separation of proteins.

• Journal of the Korean Wood Science and Technology
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• v.30 no.3
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• pp.12-17
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• 2002

The extracellular invertase (EC 3.2.1.26) (Candida utilis) preparation was obtained from the liquid medium after desalting and freeze drying. This prepared enzyme was used for the comparative purification on 4 activated matrices by liquid column affinity chromatography method. In this method there were used controlled porous glass (CPG) silanized covalently activated by keratin, silanized silica gel and silica gel covalently covered by keratin. It was found that the invertase purification process was better using both CPG matrices (silanized CPG and keratin activated CPG) than these with two silica gel supports. Also the elution coefficient of the invertase from the two CPG columns was about 93 to 94%. Two silica gel supports found to be superior in terms of purification efficiency. The invertase purification process was confirmed by PAGE electrophoresis.

• Membrane Journal
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• v.8 no.1
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• pp.50-58
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• 1998

Protein affinity membranes were prepared via coating of chitosan gel on the porous flat and hollow-fiber polysulfone membranes, followed by the immobilization of the reactive dye (Cibacron Blue 3GA) to the chitosan gel. Maximum protein binding capacity of these affinity membranes was about 70 $\mu{g/cm}^2$. Using the affinity flat membrane module, the elution chromatography of human serum albumin (HSA) was performed to determine the optimum condition of eluent buffer. The optimum condition of eluent was the universal buffer solution of 0.06 M concentration containing 1 M KCl at pH 10. For the frontal chromatography of HSA using the flat module, the dynamic protein binding capacity was rapidly decreased from the equilibrium values with increasing flow rate and HSA concentration of the loading solution. However, in the case of hollow-fiber module, the dynamic binding capacity was maintained an equilibrium value without depending on the operating conditions. These results showed that the hollow-fiber module was more effective than the flat module as an affinity chromatography column.

• Progress in Superconductivity and Cryogenics
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• v.25 no.4
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• pp.60-64
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• 2023

The manufacturing process of antibody drugs comprises two main stages: the upstream process for antibody cultivation and the downstream process for antibody extraction. The domestic bio industry has excellent technology for the upstream process. However, it relies on the technology of foreign countries to execute downstream process such as affinity chromatography. Furthermore, there are no domestic companies capable of producing the equipment for affinity chromatography. High gradient magnetic separation technology using a high temperature superconducting magnet as a novel antibody separation and purification technology is introduced to substitute for the traditional technology of affinity chromatography. A specially designed magnetic filter was equipped in the bore of the superconducting magnet enabling the continuous magnetic separation of nano-sized paramagnetic beads that can be used as affinity magnetic nano beads for antibodies. To optimize the magnetic filter that captures superparamagnetic nanoparticles effectively, various shapes and materials were examined for the magnetic filter. The result of magnetic separation experiments show that the maximum separation and recovery ratio of superparamagnetic nanoparticles are 99.2 %, and 99.07 %, respectively under magnetic field (3 T) and flow rate (600 litter/hr).

• Proceedings of the Membrane Society of Korea Conference
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• 2004.05a
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• pp.59-62
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• 2004

Affinity membranes have emerged principally to overcome the problems of limited specificity experienced with membranes that operate purely on a sieving mechanism and as an alternative to the traditional affinity resins. It is a logical expectation that affinity membranes might combine the outstanding selectivity of affinity resins with the high productivity associated with filtration membranes.(omitted)

• Journal of the Korean Chemical Society
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• v.53 no.3
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• pp.279-285
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• 2009

This paper has described about preparation of $Zr^{4+}$ affinity column based on the poly(styrene-co- gly-cidyl methacrylate) prepared by emulsion polymerization of styrene and glycidyl methacrylate in order to isolate phosphopeptide. The $Zr^{4+}$ ions were introduced after the phophonation of an epoxy group on polymeric microspheres. The successful preparation of $Zr^{4+}$-immobilized polymeric microsphere stationary phase was confirmed through Fourier transform infrared spectra, optical microscopy, scanning electron microscopy, X-ray photoelectron spectra and inductively coupled plasma-atomic emission spectrometer. The separation efficiency for $Zr^{4+}$ affinity column prepared by slurry packing was tested to phosphonated casein and dephosphonated casein. The resolution time (min) of the phosphonated casein was higher than that of dephosphated casein for $Zr^{4+}$ affinity polymeric microsphere by liquid chromatography. This $Zr^{4+}$ affinity column can be used for isolation of phosphonated casein from casein using liquid chromatography.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.316.2-317
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• 2003

Immunoglobulin Y (IgY) obtained from chicken as the immunization host brings several advantages to antibody production, such as improved yield, lower cost, longer stability, and higher specificity than mammalian immunoglobulin. In the present study, we attempted to purify Maackia fauriei lectin using antilectin IgY-affinity chromatography in order to produce a good yield and to reduce the purification time. (omitted)

• Journal of Life Science
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• v.13 no.1
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• pp.67-72
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• 2003

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and a potent immunogen. Recombinant H. pylori urease expressed in E. coli was purified by simple purification procedures utilizing (CNBr-activated Sepharose-anti-urease IgG immunoaffinity chromatography or epoxy- activated Sepharose-urea affinity chromatography. Urease was apparently bound so tightly to the anti-urease IgG resin that it could not be eluted at various elution conditions except at certain extreme pH 1, including 100 mM carbonate (pH 10.5) buffer solution, which was shown to elute slightly inactivated but relatively pure enzyme. Urease eluted from the epoxy-activated Sepharose-urea affinity column showed higher activity, but the smaller UreA subunit of the enzyme appeared as a Fainter band of diminished intensity when subjected to SDS-polyamide gel electrophoresis.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.728-732
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• 2007

In the present article, a novel fusion expression vector for Escherichia coli was developed based on the pTORG plasmid, a derivative of pET32a. This vector, named pT7MT(GenBank Accession No DQ504436), carries a T7 promoter and it drives the downstream gene encoding Metallothionein 2A(MT2A). There are in-framed multiple cloning sites(MCS) downstream of the MT2A gene. A target gene can be cloned into the MCS and fused to the C-terminal of the MT2A gene in a compatible open reading frame(ORF) to achieve fusion expression. The metal-binding capability of MT2A allows the purification of fusion proteins by metal chelating affinity chromatography, known as $Ni^{2+}$-affinity chromatography. Using this expression vector, we successfully got the stable and high-yield expression of MT2A-GST and MT2A-Troponin I fusion proteins. These two proteins were easily purified from the supernatant of cell lysates by one-step $Ni^{2+}$-affinity chromatography. The final yields of MT2A-GST and MT2A-Troponin I were 30mg/l and 28mg/l in LB culture, respectively. Taken together, our data suggest that pT7MT can be applied as a useful expression vector for stable and high-yield production of fusion proteins.

• Parasites, Hosts and Diseases
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• v.30 no.3
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• pp.191-200
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• 1992

A proteolytic enzyme was purified from the tissue extract of spargana (plerocercoids of Spirometra erinacei) by DEAE-Trisacryl M ion exchange chromatography and thiopropyl-sepharose affinity chromatography resulted in a 21-fold purification. The proteinase activity was assayed with a synthetic fluorescent substrate, carbobensoxy-phenylalanyl-7-amiso-4-trifluoromethyl-coumarin. SDS-polyacplamide gel electrophoresis of the purified materials revealed a single 28,000 dalton band. Inhibitor profiles of the band indicated that it belonged to cysteine endopeptidases. It exhibited identical pH curves with optimum at pH 5,5, and 50% activity from pH 4.7 to 8. It could completely degrade collagen chains to three identical products. It also showed some activity on hemoglobin. Furthermore, the band on immunoblots was reactive to the sera of sparganosis patients. These results suggest that the proteolytic enzyme belongs to cysteine proteinase which plays a role in the tissue penetration. Also it may be used as the antigen for diagnosis of active sparganosis.