Autor: |
Kudou M; Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-Ku, Kobe, 657-8501, Japan., Okazaki F, Asai-Nakashima N, Ogino C, Kondo A |
Jazyk: |
angličtina |
Zdroj: |
Biotechnology letters [Biotechnol Lett] 2015 Jan; Vol. 37 (1), pp. 89-94. Date of Electronic Publication: 2014 Sep 12. |
DOI: |
10.1007/s10529-014-1666-3 |
Abstrakt: |
Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes. |
Databáze: |
MEDLINE |
Externí odkaz: |
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