Expression system for synthesis and purification of recombinant human growth hormone in Pichia pastoris and structural analysis by MALDI-ToF mass spectrometry

Date

2008-01-01

Author

Çalık, Pınar
Celik, Eda
Halloran, S. Mitchell
Calik, Guezide
Ozdamar, Tuncer H.

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

142
views

0
downloads

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone (rHGH) was designed and implemented. hGH cDNA sequence was cloned into pPICZ alpha A vector under the control of AOX1 promoter, which included a polyhistidine-tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non-native Nand C-termini. Analyses of the affinity-purified rhGH product by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed a spectral peak at m/z 23699. Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa. The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus, which includes the EcoRI digestion site (Glu-Phe), the 6xHis tag for affinity purification, and the Factor Xa protease recognition sequence (Ile-Glu-Gly-Arg), a result that also indicates that the signal peptide was properly processed by P. pastoris. N-Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence. Thus, the system designed functioned with its intended purpose effectively in expression, cleavage, and purification of the recombinant product.

Subject Keywords

Biotechnology

URI

https://hdl.handle.net/11511/35930

Journal

BIOTECHNOLOGY PROGRESS

DOI

https://doi.org/10.1021/bp070294t

Collections

Department of Chemical Engineering, Article

Suggestions

OpenMETU
Core

Recombinant therapeutic protease production by Bacillus sp. Korkmaz, Nuriye; Çalık, Pınar; Department of Chemical Engineering (2007) The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5’ end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through P...
Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes Kocabıyık, Semra; Zwickl, Peter; Ozdogan, Seda (Elsevier BV, 2010-10-01) In this study we describe, the construction of a co-expression vector allowing simultaneous production of Thermoplasma volcanium 20S proteasome alpha- and beta-subunits in Escherichia coli. This heterologous expression system provided high level production of fully active 205 proteasome that can be purified easily by using a conventional two-step chromatographic technique. The recombinant proteasome was purified to homogeneity 12-fold with a specific activity of 26.5 U/mg. Sodium dodecyl sulfate-polyacrylam...
Production of recombinant proteins by yeast cells Celik, Eda; Çalık, Pınar (Elsevier BV, 2012-09-01) Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosa...
Comparison of benzaldehyde lyase production capacity in recombinant Escherichia coli and recombinant Bacillus species Kaya, Hande; Çalık, Pınar; Department of Chemical Engineering (2006) In this study, the benzaldehyde lyase (BAL, EC 4.1.2.38) production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3)...
Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species Celik, E; Çalık, Pınar (Wiley, 2004-03-01) After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) P-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH4)(2)HPO4, NH4Cl, yeast extract, casamino acids and peptone, pH, and...

Citation Formats

P. Çalık, E. Celik, S. M. Halloran, G. Calik, and T. H. Ozdamar, “Expression system for synthesis and purification of recombinant human growth hormone in Pichia pastoris and structural analysis by MALDI-ToF mass spectrometry,” BIOTECHNOLOGY PROGRESS, pp. 221–226, 2008, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/35930.