Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Abdullah, Norhafizah and Chase, Howard Allaker (2005) Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption. Biotechnology and Bioengineering, 92 (4). pp. 501-513. ISSN 0006-3592

Full text not available from this repository.

Official URL: http://dx.doi.org/10.1002/bit.20633

Abstract

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinitychromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1)for the removal of C-terminusand N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidinetagofMBP-His6 by Factor Xa and HT 15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-HiS6 to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. Oncolumn tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-1 system was superiorto the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-1 system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-1 (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-1 digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-1 digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-l for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions. © 2005 Wiley Periodicals, Inc.

Item Type:Article
Keyword:DAPase; Expanded bed adsorption; Factor Xa; Immobilized metal affinity chromatography; Removal of polyhistidine tails
Subject:Recombinant proteins
Subject:Chromatographic analysis
Subject:Protein engineering
Faculty or Institute:Faculty of Engineering
DOI Number:10.1002/bit.20633
Altmetrics:http://www.altmetric.com/details.php?domain=psasir.upm.edu.my&doi=10.1002/bit.20633
ID Code:18361
Deposited By: Azwana Abdul Rahman
Deposited On:30 Oct 2011 00:29
Last Modified:30 Oct 2011 00:29

Repository Staff Only: item control page

Document Download Statistics

This item has been downloaded for since 30 Oct 2011 00:29.

View statistics for "Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption"


Universiti Putra Malaysia Institutional Repository

Universiti Putra Malaysia Institutional Repository is an on-line digital archive that serves as a central collection and storage of scientific information and research at the Universiti Putra Malaysia.

Currently, the collections deposited in the IR consists of Master and PhD theses, Master and PhD Project Report, Journal Articles, Journal Bulletins, Conference Papers, UPM News, Newspaper Cuttings, Patents and Inaugural Lectures.

As the policy of the university does not permit users to view thesis in full text, access is only given to the first 24 pages only.