Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes

Date

2010-10-01

Author

Kocabıyık, Semra
Zwickl, Peter
Ozdogan, Seda

Metadata

Show full item record

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

Item Usage Stats

248
views

0
downloads

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-polyacrylamide gel electrophoresis revealed the presence of two unique bands of alpha-(24 kDa) and beta-(21 kDa) subunits which were combined into proteolytically active proteasome complex in vivo when they were co-expressed in E. coli. The predominant peptide hydrolyzing activity was measured with typical chromogenic substrate (Ala-Ala-Phe-pNA) for chymotrypsin-like activity. The sequence analyses of the subunit genes showed that functional domains and residues including catalytic groups are highly conserved as compared to other archaeal proteasomes.

Subject Keywords

Biotechnology

URI

https://hdl.handle.net/11511/42784

Journal

PROTEIN EXPRESSION AND PURIFICATION

DOI

https://doi.org/10.1016/j.pep.2010.05.004

Collections

Department of Biology, Article

Suggestions

OpenMETU
Core

Influence of oxygen transfer on benzaldehyde lyase production by recombinant Escherichia coli BL21(DE3) pLySs Angardi, Vahideh; Çalık, Pınar; Department of Chemical Engineering (2007) In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxy...
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)...
Exponential feeding strategy development for benzaldehyde lyase production by recombinant "Escherichia coli" Taşpınar, Hatice; Çalık, Pınar; Özdamar, Tunçer H.; Department of Biotechnology (2010) In this study, the aim was to investigate the effects of exponential feeding strategy on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21. For this purpose, the effects of medium components were investigated to optimize the initial medium composition of the fed-batch fermentations. For the batch bioreactor operations, the highest cell concentration and BAL activity were achieved in a media containing 30 g L-1 pretreated molasses, and 5 g L-1 (NH4)2HPO4 as 5.07 g L-1, and 1611 U ml-1 ...
Kinetic analyses of the effects of temperature and light intensity on growth, hydrogenm production and organic acid utilization by rhodobacter capsulatus Sevinç, Pelin; Gündüz, Ufuk; Department of Biotechnology (2010) Effects of temperature and light intensity on photofermentative hydrogen production by Rhodobacter capsulatus DSM1710 by use of acetic and lactic acids as substrates were studied. Experiments were conducted at 20, 30 and 38oC incubator temperatures under light intensities in the 1500 – 7000 lux range. pH of the medium and quantity of hydrogen forming together with quantity of biomass, and concentrations of acetic, lactic, formic, butyric and propionic acids in the medium were determined periodically. Growth...
Dynamic flux balance analysis for pharmaceutical protein production by Pichia pastoris: Human growth hormone Çalık, Pınar; Taspinar, Hatice; Soyaslan, Elif S.; Inankur, Bahar (Elsevier BV, 2011-03-07) The influence of methanol feeding rate on intracellular reaction network of recombinant human growth hormone (rhGH) producing Pichia pastoris was investigated at three different specific growth rates, namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04h(-1) (MS-0.04) where Period-I (33 <= t < 42 h) includes the early exponential growth phase; Period-II (42 <= t < 48 h) is the exponential growth phase where the specific cell growth rate decreases; Period-III (48 <= t <= 51 h) is the exponential growth phase whe...

Citation Formats

S. Kocabıyık, P. Zwickl, and S. Ozdogan, “Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes,” PROTEIN EXPRESSION AND PURIFICATION, pp. 223–230, 2010, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/42784.