Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase

Georgiou, George; Telford, John N.; Shuler, Michael L.; Wilson, David B.

doi:10.1128/aem.52.5.1157-1161.1986

Cited by 98 publications

(24 citation statements)

References 17 publications

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“…Formation of inclusion bodies (IBs) frequently occurs when heterologous proteins are expressed in Escherichia coli (1)(2)(3)(4)(5)(6)(7)(8), and recovering the recombinant protein requires refolding it into its active structure (9)(10)(11)(12). Arginine is extensively used for refolding proteins obtained from IBs and appears to be effective for a variety of proteins differing in chemical and physical properties (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24); some examples are given in this Review).…”

Section: Introductionmentioning

confidence: 99%

Role of Arginine in Protein Refolding, Solubilization, and Purification

et al. 2004

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Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria. To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution. In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells. Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage. All these arginine effects are apparently due to suppression of protein aggregation. Little is known, however, about the mechanism. Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility. The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions. In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine.

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Section: Introductionmentioning

confidence: 99%

Role of Arginine in Protein Refolding, Solubilization, and Purification

et al. 2004

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“…No a-amylase activity of E. coli[pULTV225] was detected in the culture broth or associated to cells; however, cells had a larger size and contained a high amount of intracellular inclusion bodies. This phenomenon has been reported in several cases [17], suggesting that the fusion protein is being synthesized, but cell-associated aamylase activity was not observed, probably due to the lack of processing at the leader peptide level which prevents reaching the active conformation of a-amylase. This fact has also been reported for alkaline phosphatase in E. coli [18], which is active only after processing in the periplasmic space, and for the precursor of the /3-1actamase [19].…”

Section: A Amentioning

confidence: 66%

Expression of the Streptomyces griseus α-amylase gene in Escherichia coli

Vigal¹

1994

FEMS Microbiology Letters

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The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular alpha-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no alpha-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.

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“…Such accumulation of foreign proteins may also lead to formation of inclusion bodies. Recent studies have demonstrated that inclusion bodies may be formed in both cytoplasm and periplasm, and recovery of proteins from such protein aggregates is usually difficult (Georgiou et al, 1986;Georgiou and Shuler, 1988). Since most proteins are synthesized in the cytoplasm, excretion of a target protein begins with its secretion across the cytoplasmic membrane into the periplasm.…”

Section: Methodsmentioning

confidence: 99%

“…It has been reported (Oliver, 1985) that it is due to the lack of a general support machinery for protein export in E. coli that anumber of proteins that are excreted naturally by the donor species (from which genes encoding the proteins are derived) are not excreted or excreted to only a small degree by recombinant E. coli. Although some sugars are useful for protein stabilization (Arakawa and Timasheff, 1982;Georgiou et al, 1986;Bowden and Georgiou, 1988), the requirement of the presence of these sugars at high concentrations makes their application in situ impractical. The development of alternative methodologies for in situ protein excretion in cultures of recombinant E. coli is therefore of critical importance.…”

Section: Methodsmentioning

confidence: 99%

Immobilization of Escherichia coli JM103[pUC8] in κ‐Carrageenan Coupled with Recombinant Protein Release by in Situ Cell Membrane Permeabilization

Ryan

Parulekar

1991

Biotechnology Progress

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Immobilization of Escherichia coli JM103[pUC8] was carried out with kappa-carrageenan as the support matrix. Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells. As compared to suspension cultures, much higher beta-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 (beta-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 beta-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 beta-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.

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Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase

Cited by 98 publications

References 17 publications

Role of Arginine in Protein Refolding, Solubilization, and Purification

Role of Arginine in Protein Refolding, Solubilization, and Purification

Expression of the Streptomyces griseus α-amylase gene in Escherichia coli

Immobilization of Escherichia coli JM103[pUC8] in κ‐Carrageenan Coupled with Recombinant Protein Release by in Situ Cell Membrane Permeabilization

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