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Improvements of a high-throughput protein purification process using a calcium-dependent setup.

Abstract：

:The Human Secretome Project aims to produce and purify all human secreted proteins as full-length. In order to enable this, a robust, gentle and effective purification process is needed, where multiple proteins can be purified in parallel. For this reason, a purification system based on a Protein C-tag and the HPC4 antibody with high affinity to the tag was chosen for purification. The strong binding between the tag and the antibody is specific and calcium-dependent, which allows for mild elution with EDTA. Presented here is a study comparing different protein purification base matrices coupled with the HPC4 antibody, aiming to increase the yield of purified protein and reduce the time for purification. Among the different tested matrices, Capto XP showed a high coupling degree and increased the amount of eluted protein as compared to the control matrix. By moving from batch incubation to direct sample loading and by performing the purification on the ÄKTAxpress, an automated protein purification process and a high reduction of hands-on sample handling was achieved. This new method also integrates the desalting step in the purification process, and the time for purification and analysis of each sample was decreased from five to three days. Moreover, a new mild method for matrix regeneration was developed using 50 mM EDTA pH 7.5 instead of 0.1 M glycine pH 2. This method was proven to be efficient for regeneration while maintaining the column binding performance even after nine rounds of regeneration.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Kanje S,Enstedt H,Dannemeyer M,Uhlén M,Hober S,Tegel H

doi

10.1016/j.pep.2020.105698

subject

Has Abstract

pub_date

2020-11-01 00:00:00

pages

105698

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(20)30289-8

journal_volume

175

pub_type

杂志文章

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