Recombinant murine growth hormone from E. coli inclusion bodies: Expression, high-pressure solubilization and refolding, and characterization of activity and structure

2010 ◽  
Vol 26 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Amber Haynes Fradkin ◽  
Carl S. Boand ◽  
Stephen P. Eisenberg ◽  
Mary S. Rosendahl ◽  
Theodore W. Randolph
Keyword(s):  
Growth Hormone ◽  
High Pressure ◽  
Inclusion Bodies ◽  
E Coli

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3)

2015 ◽  
Vol 110 ◽  
pp. 43-51 ◽  
Author(s):  
Sandeep Kumar ◽  
Kavish Kumar Jain ◽  
Anupam Singh ◽  
Amulya K. Panda ◽  
Ramesh Chander Kuhad
Keyword(s):  
Inclusion Bodies ◽  
Pectate Lyase ◽  
E Coli

Purification and Characterization of Recombinant sTRAIL Expressed in Escherichia coli

2004 ◽  
Vol 36 (2) ◽  
pp. 118-122 ◽  
Author(s):  
Xiao-Xia Xia ◽  
Ya-Ling Shen ◽  
Dong-Zhi Wei
Keyword(s):  
Inclusion Bodies ◽  
Tumor Cell Line ◽  
Single Step ◽  
Dilution Method ◽  
High Concentration ◽  
Purification And Characterization ◽  
E Coli ◽  
Metal Affinity ◽  
Soluble Trail

Abstract As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has drawn considerable attention. This report presented the purification and characterization of soluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized and refolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purified to electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purified sTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with β-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.

scholarly journals PRODUKSI PROTEIN REKOMBINAN HORMON PERTUMBUHAN IKAN KERAPU

2012 ◽  
Vol 7 (2) ◽  
pp. 231
Author(s):  
Irvan Faizal ◽  
Ratu Siti Aliah ◽  
Muhammad Husni Amarullah ◽  
Novi Megawati ◽  
Sutanti Sutanti ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Inclusion Bodies ◽  
Recombinant Growth Hormone ◽  
E Coli

Salah satu spesies ikan yang menjadi target produksi perikanan budidaya nasional adalah ikan kerapu tikus (Cromileptes altivelis). Ikan kerapu tikus merupakan ikan laut budidaya komoditas ekspor, namun laju pertumbuhannya sangat lambat. Oleh karena itu, perlu dilakukan usaha budidaya yang mampu menaikkan laju pertumbuhan ikan kerapu tikus. Pendekatan nutrisi melalui penggunaan hormon pertumbuhan (Growth Hormone, GH) pada usaha budidaya diyakini mampu meningkatkan kecepatan tumbuh ikan budidaya. Pada penelitian ini, melalui teknologi DNA rekombinan telah dilakukan isolasi dan identifikasi gen GH yang selanjutnya dilakukan produksi protein rekombinan GH (recombinant Growth Hormone, rGH) dengan memanfaatkan penggunaan bakteri E. coli. Dalam pelaksanaan penelitian dilakukan konstruksi rGH yang menghasilkan bakteri E. coli BL21 (DE3) yang mampu memproduksi protein rGH. Produksi rGH dilakukan pada skala bioreaktor. Proses isolasi produk rGH-nya dalam bentuk pellet inclusion bodies yang selanjutnya dicampur dengan pelet pakan komersil hingga konsentrasi akhir protein dalam pakan mencapai 1 ng/6 mg pakan, di mana setelah dikering-anginkan, pelet pakan protein rekombinan GH dapat diaplikasikan untuk budidaya ikan kerapu tikus dan ikan budidaya lainnya.

scholarly journals PRODUCTION AND CHARACTERIZATION OF RECOMBINANT PROTEINS CONTAINING BET V 2 ANTIGENIC EPITOPES OF BETULA PENDULA

2012 ◽  
Vol 9 (6) ◽  
pp. 28-31
Author(s):  
A V Chernysheva ◽  
V V Tyutyaeva ◽  
A V Pivovarova ◽  
I V Andreev ◽  
M N Sankov ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Inclusion Bodies ◽  
Birch Pollen ◽  
Elisa Test ◽  
Recombinant Allergen ◽  
Bacterial Cells ◽  
E Coli ◽  
Immunological Tests ◽  
Linear Epitopes

Aim of investigation. Production of immunologically active recombinant protein of Bet v 2 allergen ofbirch pollen. Materials and methods. mRNA was isolated from a sample ofbirch pollen. cDNA library was derived using SMART technology. Gene Bet v 2 was amplified by means of PCR with primers from the cDNA. The resulting PCR fragment of the gene was cloned into the vector pET29b(+). The recombinant protein Bet v 2 was expressed in cells E. coli, transformed with a plasmid. The recombinant protein was purified using NiNTA agarose. Immunological activity of the recombinant protein Bet v 2 was measured by ELISA and immunoblot methods. Results. The production system of the recombinant allergen Bet v 2 preparation suitable for immunological tests was developed during the research project. In the first phase allergen Bet v 2 gene was cloned from birch pollen collected in Russia. The gene was inserted into the vector pET29(+) for expression in bacterial cells. The expression cell strain E. coli was obtained with this plasmid. The synthesis of the recombinant protein that accumulates in inclusion bodies was activated in bacterial cells. The procedure of recombinant protein Bet v 2 isolation from inclusion bodies was developed by one round of chromatography purification. The recombinant protein isolation was carried out by chromatography on Niagarose. The highly purified preparation of the recombinant allergen was obtained as a result. The recognition of the recombinant protein Bet v 2 by sera varied in ELISA, indicating a different degree of patients sensitization to this allergen. In the immunoblot test the preparation was active only in 15% of cases. Apparently, reactive epitopes of the allergen are mainly conformational ones and are active in ELISA test, whereas linear epitopes, that are active in immunoblot, are in the minority. Conclusion.The system for production of recombinant allergen Bet v 2 preparation suitable for immunological tests has been developed.

scholarly journals Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247689
Author(s):  
Bastian Heim ◽  
René Handrick ◽  
Marcus D. Hartmann ◽  
Hans Kiefer
Keyword(s):  
G Protein ◽  
Inclusion Bodies ◽  
Protein Unfolding ◽  
G Protein Coupled Receptors ◽  
Orphan Receptor ◽  
Cubic Phase ◽  
E Coli ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

2000 ◽  
Vol 347 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Sanya J. SANDERSON ◽  
Kevin G. J. POLLOCK ◽  
James D. HILLEY ◽  
Morten MELDAL ◽  
Phaedria ST HILAIRE ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Cysteine Proteinase ◽  
Native Enzyme ◽  
Leishmania Mexicana ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Mature Enzyme ◽  
Pro Region

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8∆CTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded > 3.5 mg of active enzyme per litre of E. coli culture.

Expression, Purification and Characterization of Amantadine Receptor in Escherichia coli

2012 ◽  
Vol 161 ◽  
pp. 88-93 ◽  
Author(s):  
Hai Xin Sun ◽  
Li Min Cao ◽  
Hong Lin ◽  
Fang Lv
Keyword(s):  
Inclusion Bodies ◽  
Equilibrium Dialysis ◽  
Molecular Size ◽  
M2 Protein ◽  
Purification And Characterization ◽  
E Coli ◽  
His Tag ◽  
Sds Page ◽  
Fast Flow

In order to obtain large quantities of broadly selective receptor as one diagnose agent to detect amantadine residue, the M2 protein gene with a His-tag was ligated into pET11a and transferred into E. coli BL21 (DE3) cell. The recombinant E. coli was cultured in liquid LB culture. SDS-PAGE result showed the recombinant M2 protein (rM2) was expressed as insoluble inclusion bodies with about 18KDa in molecular size. rM2 protein was further recognized by Western blot and purified by Ni Sepharose 6 Fast Flow and then refolded. The equilibrium dialysis result showed the rM2 protein had the binding constant of 1.1×105, and stoichiometry of 4.2. The above result showed the rM2 has the potential as biological diagnose agent to the detection of amantadine residue.

scholarly journals Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli

1993 ◽  
Vol 295 (3) ◽  
pp. 719-724 ◽  
Author(s):  
A Igout ◽  
J Van Beeumen ◽  
F Frankenne ◽  
M L Scippo ◽  
B Devreese ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Hormone ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Expression System ◽  
Pituitary Hormone ◽  
Native Form ◽  
E Coli ◽  
Placental Growth Hormone

The hGH-V (or hGH-2) gene codes for human placental growth hormone (hPGH). Secretion of hPGH is continuous, in contrast with the pulsed secretion of pituitary growth hormone (hGH) which it progressively replaces in the maternal bloodstream. hGH-V cDNA has previously been cloned and isolated. Analysis of its nucleotide sequence has revealed a 191-residue protein, hPGH, differing from hGH at 13 positions. The calculated pI is more basic than that of the pituitary hormone. Here we have inserted hGH-V cDNA into the pIN-III-ompA3 plasmid in order to produce hPGH in its native form in Escherichia coli D1210. Expression of hGH-V cDNA in E. coli is significantly lower than that of hGH cDNA with the same expression system. The hPGH produced in E. coli was purified in quantities sufficient to allow its biochemical and immunochemical characterization. The molecular mass of the protein was determined by electrospray m.s. The determined mass, 22,320 Da, agrees well with the molecular mass calculated from the translated cDNA sequence, assuming the presence of two disulphide bridges. Having established the technique for producing hPGH with a primary structure identical to the natural, non-glycosylated, 22 kDa isoform, we can now plan the full physicochemical and pharmaceutical characterization of this new hormonal entity.

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