Research in Molecular Medicine
Research in Molecular Medicine
Res Mol Med (RMM)
Medical Sciences
http://rmm.mazums.ac.ir
1
admin
2322-1348
2322-133X
10.29252/rmm
en
jalali
1396
5
1
gregorian
2017
8
1
5
3
online
1
fulltext
en
Expression and Purification of Soluble form of Human Parathyroid Hormone (rhPTH1-34) by Trx Tag in E. coli
بیوتکنولوژی
Biotechnology
پژوهشي
Research
<div style="text-align: justify;"><strong>Background:</strong> Parathyroid Hormone (PTH) is secreted by parathyroid glands and controls the level of calcium in bones and kidney. PTH is a small polypeptide with 84 amino acids, but the first 34 amino acids of which are enough for hormone biological activity and can be used in the treatment of Osteoporosis. The expression efficiency of recombinant human parathyroid hormone rhPTH (1-34) or Teriparatide using a cleavable fusion protein strategy was compared in two strains of <em>E. coli.</em><br>
<strong>Materials and Methods:</strong> A cassette was designed and fully synthesized for prokaryotic expression of rhPTH using pET system. From 5’ to 3’, the cassette consisted of: Trx tag to increase the solubility of protein, His tag for purification and detection of protein, enterokinase site to cleave all fusion moieties, and an optimized gene code for Teriparatide corresponding to the amino acid sequence of hPTH. This cassette was cloned into pET32a vector. The vector was simultaneously transformed and expressed in two different <em>E. coli</em> strains. The ability of strains for expression of this recombinant pharmaceutical was compared. Early expression was confirmed by SDS-PAGE and Western Blotting. The soluble fusion protein was harvested and purified by immobilized affinity chromatography. Then the fusion moiety was released from Teriparatide by enterokinase digestion.<br>
<strong>Results:</strong> The fusion form of rhPTH was efficiently expressed in both <em>E. coli</em> strains. However, the percentage of the target protein to the total protein content in Rosetta-gami was more than its amount in BL21 (­60 % vs 25%).The fusion protein was highly purified with Ni-NTA column. Up to 18.5 mg/ml of pure fusion protein has been obtained from 1-liter Rosetta-gami strain of <em>E. coli</em>. The pure Teriparatide was released by enterokinase digestion.<br>
<strong>Conclusion: </strong>The pure rhPTH (1-34) produced here, could be the subject for biological activity and quality control assessments, and following formulation processing, it could be applied as a peptide drug in the treatment of Osteoporosis.<br>
</div>
Enterokinase, Fusion protein, parathyroid hormone, BL21, Rosetta-gami
26
31
http://rmm.mazums.ac.ir/browse.php?a_code=A-10-914-1&slc_lang=en&sid=1
Sanaz
Yari
sanazyari1991@gmail.com
10031947532846006775
10031947532846006775
No
Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
پژوهشکده ی فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران، ایران
Farida
Behzadian
fbehzadian@yahoo.com
10031947532846006776
10031947532846006776
Yes
Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
پژوهشکده ی فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران، ایران
Hamideh
Rouhani nejad
حمیده
روحانی نژاد
rohaninejhad@gmail.com
10031947532846006777
10031947532846006777
No
Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
پژوهشکده ی فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران، ایران
Mohammad reza
Masoumian
محمد رضا
معصومیان
masoumian.mr@gmail.com
10031947532846006778
10031947532846006778
No
Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
پژوهشکده ی فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران، ایران
Mahdi
Karimi
مهدی
کریمی
karimi_mhi92@yahoo.com
10031947532846006779
10031947532846006779
No
Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
پژوهشکده ی فناوری زیستی، دانشگاه صنعتی مالک اشتر، تهران، ایران