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At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids. Opposite to the wild type, the cardiolipin-depleted membrane is more fragile with respect to sonication and osmotic chock, at severe limitation, and results in extensive foaming during the process. Protein leakage and cell lysis is, however, lower in the mutant most likely due to the increased amounts of saturated fatty acids, which might be a possible strategy to overcome the reduced amounts of membrane-strengthening cardiolipin. The membrane potential of the outer surface is negative, however less negative for the mutant. This was supported by aqueous two-phase extraction experiments which, furthermore indicated a difference in outer surface hydrofobicity. These findings suggest that the relA1 gene has a defined, but ppGpp-independent, role in cells with a slowly decreasing metabolism of glucose to control the membrane morphology.

A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 l to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO(50)PO(50) (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)(4) tag was used for enhanced target protein partitioning to the EO(50)PO(50) phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO(50)PO(50) phase from the first step was supplemented with a non-ionic detergent (C(12-18)EO(5)) and heated to the cloud point (CP) temperature (45 degrees C). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.

In vivo proteolysis of protein ZZT0, derived from the B domain of staphylococcal protein A, was investigated in Escherichia coli before and after insertion of 1-3 multiples of the tetrapeptide Ala-Trp-Trp-Pro close to the C-terminus of ZZT0. Before insertion, ZZT0 was proteolytically stable as judged from the purity of IgG binding proteins up to 1 h after inhibition of protein synthesis with chloramphenicol. Insertion of 1-3 units of Ala-Trp-Trp-Pro into ZZT0 increased progressively the sensitivity to proteolysis and induced DnaK and GroEL binding to the protein. The time for 50% in vivo hydrolysis of the full length protein derivative that was most susceptible to proteolysis, i.e. with three tetrapeptide units, was about 40 min when cultivated in a bioreactor and about 4 min in a shaken flask culture. Molecular masses and N-terminal sequences of the main degradation products indicated that protein ZZT0 is cleaved at identical sites irrespective of the number of inserted tetrapeptide units and that the cleavage sites are located far from the insertion point. Insertion of another hydrophobic amino acid, isoleucine, as the tetrapeptide Ala-Ile-Ile-Pro, only induced a slight proteolysis of the ZZT0 molecule under similar conditions. This indicates that the insertion of tryptophan residues, rather than of a general hydrophobic segment, plays an essential role in the induced proteolysis of the ZZT0 protein. [ABSTRACT FROM AUTHOR]

Abstract: In this work, the interfacing of a poly(ethylene glycol) (PEG)–phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan–proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)4 was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K–Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to >95% purity according to silver stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels with a total yield of 83% during the two-step recovery process. [Copyright &y& Elsevier]