(44.201.94.72)
Users online: 3962    [ij] [ij] [ij] 
Email id
 

Current Trends in Biotechnology and Pharmacy
Year : 2010, Volume : 4, Issue : 1
First page : ( 568) Last page : ( 577)
Print ISSN : 0973-8916. Online ISSN : 2230-7303.

High Yield Expression of Human Recombinant PTH (1–34)

Gangireddy Srinivasa R.1,2, Madhavi Radha D.1, Ravikanth Kosana R.1, Reddy Praveen K.1, Konda Venkat R.1, Rao K R S Sambasiva2,*, Gunwar Sripad1

1Virchow Research Institute, Hyderabad, 500055, Andhra Pradesh, India

2Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510, Andhra Pradesh, India

*For Correspondence -krssrao@yahoo.com

Abstract

Human parathyroid hormone (hPTH), synthesized in the parathyroid gland as a linear peptide, is one of the key regulatory molecules in calcium homeostasis and bone resorption. The N terminus region of hPTH (1–34) is a functionally important part of the molecule that is sufficient and necessary for potently executing most of the hormonal actions. Therefore, hPTH (1–34) is considered to be an attractive therapeutic agent in the treatment of osteoporosis. Here, we describe a high yield expression and purification method for the production of hPTH (1–34) from Escherichia coli. The hPTH (1–34) was expressed as a fusion protein in soluble form, and found to be more than 25% of total proteins. The fusion protein was first purified by GST affinity chromatography and, after cleavage of GST with Factor Xa, the peptide (hPTH 1–34) was further purified by reversed phase Source-30™ chromatography. This double purification strategy produced 30mg/l of hPTH (1–34) with purity = 98%. The identity of the purified peptide was confirmed by mass spectrometry and N-terminal sequencing analysis. The biological activity of the peptide was confirmed in the rat osteogenic cell line UMR-106 by measuring cAMP levels, which were identical to hPTH standards, indicating that purified rhPTH (1–34) has full biological activity.

Top

Keywords

Factor Xa, Glutathione S transferase, Protein purification, Recombinant human parathyroid hormone.

Top

 
║ Site map ║ Privacy Policy ║ Copyright ║ Terms & Conditions ║ Page Rank Tool
565,370,726 visitor(s) since 30th May, 2005.
All rights reserved. Site designed and maintained by DIVA ENTERPRISES PVT. LTD..
Note: Please use Internet Explorer (6.0 or above). Some functionalities may not work in other browsers.