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Immobilization of Escherichia coli JM103[pUC8] in κ‐Carrageenan Coupled with Recombinant Protein Release by in Situ Cell Membrane Permeabilization
Author(s) -
Ryan Wen,
Parulekar Satish J.
Publication year - 1991
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp00008a004
Subject(s) - ethylenediaminetetraacetic acid , escherichia coli , plasmid , cell culture , recombinant dna , extracellular , chemistry , cell , microbiology and biotechnology , biology , membrane , biochemistry , chelation , gene , genetics , organic chemistry
Immobilization of Escherichia coli JM103[pUC8] was carried out with κ‐carrageenan as the support matrix. Substantial natural excretion of β‐lactamase, attributable to the less intact membrane of plasmid‐harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the β‐lactamase produced was retained in the cells. As compared to suspension cultures, much higher β‐lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid‐bearing cells (improved plasmid stability) were observed in immobilized cell cultures. Further enhancement in excretion of the recombinant protein (β‐lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA). While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA. Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes. Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of β‐lactamase when compared to the enzyme production in EDTA‐free cultures. More frequent addition of EDTA increased the period of retention of plasmid‐bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of β‐lactamase. Improvement in plasmid stability in EDTA‐treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid‐free and plasmid‐bearing cells, since plasmid‐free cells were subject to more reduction in specific growth rate than were plasmid‐bearing cells.