Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions

Microbial Cell Factories
Jinping Du, Bernd H A Rehm

Abstract

Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IF...Continue Reading

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Citations

Nov 11, 2017·Biotechnology Letters·Jinping Du, Bernd H A Rehm
Mar 3, 2020·Frontiers in Bioengineering and Biotechnology·Jin Xiang WongBernd H A Rehm
Apr 2, 2018·Biotechnology Letters·Xiaofeng YangZhanglin Lin
Mar 21, 2020·Frontiers in Bioengineering and Biotechnology·Jin Xiang WongBernd H A Rehm

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Methods Mentioned

BETA
ELISA
X-ray
circular dichroism

Software Mentioned

IMAGE LAB

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