PRODUCTION OF RECOMBINANT HUMAN GROWTH HORMONE IN ESCHERICIA COLI STRAIN ORIGAMI AND COMPARING FREEZE/ THAW AND SONICATION METHODS FOR PROTEINS EXTRACTION

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SAVARI MARZIEH; ZARKESH ESFAHANI SAYYED HAMID

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Journal: BIOLOGICAL JOURNAL OF MICROORGANISM Year:2015 | Volume:4 | Issue:14 Page(s): 141-152

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Title

PRODUCTION OF RECOMBINANT HUMAN GROWTH HORMONE IN ESCHERICIA COLI STRAIN ORIGAMI AND COMPARING FREEZE/ THAW AND SONICATION METHODS FOR PROTEINS EXTRACTION

Author(s)

SAVARI MARZIEH | ZARKESH ESFAHANI SAYYED HAMID | Issue Writer Certificate

Keywords

ORIGAMI (DE3)Q2

RECOMBINANT HUMAN GROWTH HORMONEQ2

ULTRASONICQ1

FREEZE/ THAWQ2

DOT BLOTQ2

WESTERN BLOTQ2

Abstract

Introduction: Human growth hormone (hGH) is a single-chain polypeptide derived from pituitary gland that participates in a wide range of biological functions and has therapeutic applications. The hormone consists of 191 amino acid residues (22kD) which folds into a four-helix bundle structure with two disulfide bridges. Due to the complication of internal cell protein extraction, several methods have been established for this purpose that most of them need special instruments. Thus, in the preliminary studies, scientists need a quick and simple method for cell disruption and protein extraction. The aim of the present study was production of hGH in E.coli and comparison between the freeze/thaw and Sonication method for protein extraction from cells.Materials and methods: The competent cells of E. coli Origami (DE3) were transformed using plasmid containing human growth hormone gene and then cultured in LB medium. After IPTG induction, the hGH was extracted using various methods including bacterial lysing, freeze/thaw and sonication methods and then extracted protein was assessed using ELISA, Bradford, DOT BLOT and WESTERN BLOTting.Results: According to our findings, bacterial transformation showed high efficiency of transformation. Furthermore, the results of DOT BLOT and WESTERN BLOT showed production of recombinant hGH at high levels. Protein extraction measurement using ELISA and Bradford methods indicated that the proportion of the extracted protein in the slow freeze/thaw method was higher than the fast freeze/thaw method and was almost equal to sonication method.Discussion and conclusion: The extracted protein levels in both of the slow FREEZE/ THAW and ULTRASONIC methods was equal, which introduces the slow freeze/thaw method as an excellent substitution for ULTRASONIC method when special instruments are not available.

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APA: Copy

SAVARI, MARZIEH, & ZARKESH ESFAHANI, SAYYED HAMID. (2015). PRODUCTION OF RECOMBINANT HUMAN GROWTH HORMONE IN ESCHERICIA COLI STRAIN ORIGAMI AND COMPARING FREEZE/ THAW AND SONICATION METHODS FOR PROTEINS EXTRACTION. BIOLOGICAL JOURNAL OF MICROORGANISM, 4(14), 141-152. SID. https://sid.ir/paper/237355/en

Vancouver: Copy

SAVARI MARZIEH, ZARKESH ESFAHANI SAYYED HAMID. PRODUCTION OF RECOMBINANT HUMAN GROWTH HORMONE IN ESCHERICIA COLI STRAIN ORIGAMI AND COMPARING FREEZE/ THAW AND SONICATION METHODS FOR PROTEINS EXTRACTION. BIOLOGICAL JOURNAL OF MICROORGANISM[Internet]. 2015;4(14):141-152. Available from: https://sid.ir/paper/237355/en

IEEE: Copy

MARZIEH SAVARI, and SAYYED HAMID ZARKESH ESFAHANI, “PRODUCTION OF RECOMBINANT HUMAN GROWTH HORMONE IN ESCHERICIA COLI STRAIN ORIGAMI AND COMPARING FREEZE/ THAW AND SONICATION METHODS FOR PROTEINS EXTRACTION,” BIOLOGICAL JOURNAL OF MICROORGANISM, vol. 4, no. 14, pp. 141–152, 2015, [Online]. Available: https://sid.ir/paper/237355/en

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