Recombinant Escherichia coli strain produces a ZZ domain displaying biopolyester granules suitable for immunoglobulin G purification

Abstract

The immunoglobulin G (IgG) binding ZZ domain of protein A from Staphylococcus aureus was fused to the N terminus of the polyhydroxyalkanoate (PHA) synthase from Cupriavidus necator. The fusion protein was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and mediated formation of ZZ domain-displaying PHA granules in recombinant Escherichia coli. The IgG binding capacity of isolated granules was assessed using enzyme-linked immunosorbent assay and could be enhanced by the overproduction of the ZZ-PHA synthase. ZZ-PHA granules enabled efficient purification of IgG from human serum.

FIG. 1.

ELISA using various PHA granules and anti-IgG antibodies for the detection of IgG bound to PHA granules. PHA granules were isolated from recombinant E. coli harboring various plasmids. Plasmids contained either the lac promoter or the T7 promoter for gene expression. The following versions of the PHA synthase mediated production of PHA granules: WT, wild-type PHA synthase; ZZ(−), ZZ-PHA synthase without signal peptide; ZZ(+), ZZ-PHA synthase plus signal peptide. Goat polyclonal anti-human IgG-horseradish peroxidase conjugates were used for detection of bound human IgG. Equal amounts of PHA granule protein (0.37 μg), corresponding to 2.6 μg polyhydroxybutyrate, were added to each well. Measurements were conducted in quadruplicate, and the mean value and the standard deviation are indicated.

FIG. 2.

SDS-PAGE analysis of proteins bound in vitro to either ZZ-PHA granules or protein A-Sepharose and released after elution. Lanes: M, molecular weight standard; 1, human serum; 2, proteins eluted from protein A-Sepharose beads; 3, proteins eluted from wild-type PHA granules; 4, proteins eluted from ZZ-PHA granules displaying the ZZ domain without signal sequence. The heavy and light chains of IgG are indicated.