• Archives of Pharmacal Research
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• 제14권3호
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• pp.231-236
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• 1991

The high affinity binding sites for angiotensin II were solubilized from rat liver membranes by treatment with CHAPS. The binding protein was also partially purified by angiotensin III inhibitor-coupled Affi-gel affinity chromatography. Binding to the intact membrances as well as to the solubilized preparation was specific and saturable. According to the Scatchard plot, the membrane preparations exhibited a single class of high affinity binding sites with a Kd OF 0.71 nM. The solubilized preparation also showed the presence of a single class of bindings sites with less affinity (Kd of 14 nM). Meanwhile the competition studies using angiotensin II analogues represented two separate binding sites for angiotensin II and single binding site for antagonist. These latter findings were correlated to the results provided by Garrison's research group. More works are needed to clarify this discrepancy.

• 대한의생명과학회지
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• 제18권2호
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• pp.96-103
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• 2012

Kv 4.2 ion channel protein has an ability to open at subthreshold membrane potentials and to recover quickly from inactivation. That is very important for neuronal signal transmission in vertebrate brain. In order to purify Kv 4.2 protein, the novel purification methods were experimented. The purification procedure utilized chromatography on DE-52 ion exchange column and affinity chromatography on a WGA-Sepharose 4B, and Kv 4.2 affinity column chromatography. It was found that 0.5% (wt./vol.) Triton X-100 detergent in lysis buffer worked well for Kv 4.2 protein solubilization from rat brain membrane. Protein quantitative determination was conducted by BCA method at 562 nm for each purification step to avoid determination interference of protein at 280 nm by detergent. The confirmation of Kv 4.2 existence and amount is performed using by SDS-PAGE/immunoblotting or 96-well dot blotting. The Kv 4.2 without interacting protein that contains carbohydrate, was purified from novel biochemical 3-steps purification method for further research.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.62-72
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• 2001

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 has been successfully cloned and expressed in Escherichia coli. Expression of D-carbamoylase gene under the 17 promoter in different host strains showed that the optimal expression was achieved in E. coli JM109 (DE3) with a 9-fold increase in enzyme production compared to the wild-type strain. The co-expression of the GroEL/ES protein with D-carbamoylase protein caused an in vivo solubilization of D-carbamoylase in an active form. The synergistic effect of GroEL/ES at 28$^{\circ}C$ led to 60 ％ solubilization of the total expressed target protein with a 6.2-fold increase in enzyme activity in comparison to that expressed without GroEL/ES and 43-fold increase in enzyme activity compared to A. tumefaciens AM 10. Attempts to express D-carbamoylase in an altered redox cytoplasmic milieu did not improve the enzyme production in an active form. The Histidyl-tagged D-carbamoylase was purified in a single step by Nickel-affinity chromatography and was found to have a specific activity of 9.5 U/mg protein.

• 한국미생물·생명공학회지
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• 제22권1호
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• pp.1-6
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• 1994

We have studied the relation between Ca$^{2+}$-ATPase and trigger peptidase(TPase) which are membeane protein well known as their significant role for signal transduction of mating pheromone in heterobasidiomycetous yeast. Rhodosporidium toruloides. We found out that there were Ca $^{2+}$-ATPase and TPase together in isolated calmodulim binding protein(CBP), usion calmodulin affinity column chromatography after solubilization of mation type a cell membrane protein, and that the dependence of enzyme activity of both the enzymes on Ca$^{2+}$, phospholipid and nonionic detergent are similar. However, Ca$^{2+}$-ATPase hed quite absolute dependence on calmodulin and, on the other hand, TPase didn't have any dependence. Judging from the fact that there are both enzymes in CBP which the dependence of calmodulin are quite different, we found out that both enzymes were made to their compound and existed in mating type a cell membrane.

• Korean Chemical Engineering Research
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• 제55권3호
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• pp.369-378
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• 2017

혈관 내피세포 성장인자(Vascular endotherial growth factor, VEGF)는 혈관 투과율 조절이나 혈관 생성에 관련된 단백질로 임상용으로 쓰일 가능성이 높다. 이 단백질은 고순도와 고효율로 상업적으로 대량생산이 필요하다. 유비퀴틴 융합 단백질로 온화한 조건에서 용해시키기 위해 다양한 조건을 연구하였고, pH와 변성제 변화를 시도하였다. BL21 (DE3) 대장균 숙주세포에서 pET28-a 벡터를 사용하여 재조합 대장균을 제조하여, 20 L의 회분식 배양으로 14 g/L농도의 세포배양을 하였다. 발효 후UBP1 효소 분해와 재접힘 단계를 포함한 4단계의 크로마토그래피 공정으로 구성된 정제공정으로 VEGF를 정제하였다. 유비퀴틴 융합단백질로 2 M 요소와 pH10 온화한 조건에서 VEGF의 정제가 가능하였다. 2번의 Ni-affinity 크로마토그래피컬럼을 이용하여 고효율의 재접힘과 이합체화 공정을 수행하였다. DEAE (Diethyl Amino Ethyl) 음이온 교환 컬럼을 통하여 변형체(multimeric, misfolded)단백질과 endotoxin을 제거 할 수 있었다. 젤 여과 크로마토그래피를 이용하여 dimer와 monomer를 분리 하여 이합체화 VEGF를 제조하였다. 최종 VEGF의 특성분석을 SDS-PAGE (Soidum Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) 전기영동, RP-HPLC (Reversed Phase High Performance Liquid Chromatography)으로 하여 순도 97% (RP-HPLC기준)를 얻었다.

• Biomolecules & Therapeutics
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• 제1권2호
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• pp.188-195
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• 1993

In order to understand the machanism of action and regulation of ${\beta}$-adrenergic receptor in terms of molecular level, the purification of receptor protein has a fundamental importance. Moreover, species differences among avian, amphibian and mammalian ${\beta}$-adrenergic receptors make it more important to purify mammalian ${\beta}$-adrenergic receptor. Because ${\beta}$-adrenergic receptor is an integral membrane protein, it must be solubilized from the membrane for the purification. The purpose of the present study was to solubilize and characterize the mammalian $\beta$-adrenergic receptor from guinea pig lung in quantities by more efficient and practical method eventually to purify receptor. Guinea pig lung membrane preparation was solubilized by sequential treatment of buffers containing low and high concentration of digitonin which are 0.2 and 1.2% respectively. About 50% of the total receptor pool was released by this double extraction procedure. The $\beta$-adrenoceptors in the digitonin extract were identified using the ${\beta}$-adrenergic antagonist, (-)-[$^3H$]-dihydroalprenolol ([$^3H$]DHA). The solubilized receptor retained all of the essential characteristics of membrane-bound receptor, namely saturability; stereoselectivity; high affinity to ${\beta}$-adrenergic drugs. For the measurement of soluble receptor activity, Sephadex G-50 chromatography method has been widely used. Inspite of its accuracy and wide acceptance, this technique employed troublesome column work which required long time to assay the activity of receptor. We employed another methods to measure receptor activity. When using 0.5% polyethylenimine pretreated GF/B glass fiber filter, filtration technique could be used to measure soluble receptor activity. This technique enabled us to reduce the total amount of time to assay by a factor of 4 as well as to detect soluble receptor. In the present study, we could establish more efficient and practical solubilization method of mammalian $\beta$-adrenergic receptor. The rapidity and high yield of this solubilization scheme, together with the favorable recovery of the receptor activity, are significant steps toward the ultimate purification of the mammalian $\beta$-adrenergic receptor. The result of this study together with more convenient purification method could provide large amount of purified receptor with ease for various research purposes.