Degradation of secreted proteins in Escherichia coli.
Explore the source record for details and available documents.
Blotting, Western↗
Biomedical subjects
G Georgiou
Publications and source records attributed to G Georgiou.
Transport and anchoring of beta-lactamase to the external surface of Escherichia coli.
The outer membrane of Gram-negative bacteria presents an effective barrier that restricts the release of proteins from the cell. Virtually all extracellular proteins of Gram-negative bacteria are exported by specialized systems requiring the action of several gene products. We have constructed a tripartite fusion consisting of (i) the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein, (ii) amino acids 46-159 of the outer membrane protein OmpA, and (iii) the complete mature beta-lactamase (EC 3.5.2.6) sequence. This protein had an enzymatically active beta-lactamase and was found predominantly in the outer membrane. Immunofluorescence microscopy, the accessibility of the fusion protein to externally added proteases, and the rates of hydrolysis of nitrocefin and penicillin G by whole cells demonstrated that a substantial fraction (20-30%) of the beta-lactamase domain of the fusion protein was exposed on the external surface of E. coli. In cells grown at 24 degrees C the localization of beta-lactamase on the cell surface was almost quantitative (greater than 80% of the enzymatically active protein was exposed to the extracellular fluid) as determined by nitrocefin and penicillin G hydrolysis and trypsin accessibility. These results demonstrated that a soluble protein, beta-lactamase, can be transported through--and become anchored on--the outer membrane by fusion to the proper targeting and localization signals.
Amino Acid Sequence↗
Surface-active compounds from microorganisms.
Microbial surfactants are a structurally diverse group of compounds consisting of hydrophilic and hydrophobic domains and which partition preferentially at interfaces. Biosurfactants are of increasing interest commercially as substitutes for synthetic surfactants particularly for environmental applications. This article discusses recent progress in the genetic and biochemical analysis of biosurfactant synthesis as well as the current status of fermentation technologies.
Bacteria↗
Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide.
beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.
Ampicillin↗
Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli.
The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A-beta-lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5 less than or equal to pH less than or equal to 6.0) and a low temperatures. These conditions were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn2+ ions.
Bacterial Proteins↗
Structure and morphology of protein inclusion bodies in Escherichia coli.
We have studied the structure and characteristics of inclusion bodies formed by the enzyme beta-lactamase in the periplasmic space of Escherichia coli or in the cytoplasm, following expression of the protein without its signal sequence. Electron microscopy of highly purified protein aggregates using a novel sucrose gradient centrifugation procedure revealed striking morphological differences. Periplasmic inclusion bodies were essentially amorphous whereas the protein particles in the cytoplasm were highly regular. Depending on the cellular location, the inclusion bodies exhibited differences in protein composition even though they were formed by the expression of the same polypeptide chain. It was shown that the chaperonins GroEL and SecB are not incorporated into the inclusion bodies. Furthermore, the degree of solubilization of the inclusion bodies in the presence of denaturants and the sensitivity of the aggregated proteins to protease digestion indicated that the differences between cytoplasmic and periplasmic inclusion bodies extend to the conformation of the associated polypeptide chains.
Cell Compartmentation↗
Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo.
Protease III, the product of the ptr gene, is a 110-kDa periplasmic protease with specificity towards insulin and other low-molecular-weight substrates (less than 7,000 molecular weight) in vitro (Y.-S.E. Cheng and D. Zipser, J. Biol. Chem. 254:4698-4706, 1979). Escherichia coli strains deficient in protease III were constructed by insertional inactivation of the ptr gene. This mutation did not appear to affect the function of the adjoining recB and recC genes. Expression of protein A-beta-lactamase, a protease-sensitive secreted polypeptide, was increased approximately twofold in ptr cells. A comparable increase in the half-life of protein A-beta-lactamase was observed by pulse-chase experiments, suggesting that protease III is involved in the catabolism of high-molecular-weight substrates in vivo, ptr mutants exhibited no detectable phenotypic alterations except for a slight reduction in growth rate. When the ptr mutation was transferred to a strain deficient in the secreted protease DegP, a further decrease in growth rate, as well as an additive increase in the expression of the fusion protein, was observed. A ptr degP ompT mutant strain resulted in a further increase in expression in minimal medium but not in rich medium.
Blotting, Southern↗
Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli.
High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli. The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm. With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate. Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein. Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis. These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway. Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G. (1988) Biotechnol. Prog. 4, 97-101). We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing. It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro.
Cytosol↗
Affinity immobilization of a genetically engineered bifunctional hybrid protein.
Protein A from Staphylococcus aureus (SpA) is a receptor for the Fc domain of several classes of antibodies including immunoglobin G (IgG). A hybrid protein consisting of protein A and the enzyme beta-lactamase has been constructed using recombinant DNA techniques. The functional characteristics of the hybrid protein adsorbed on IgG-coated Sepharose matrices were studied in detail and compared to those of (i) the hybrid protein in solution and (ii) beta-lactamase covalently immobilized on CNBr-activated Sepharose. Protein A--beta-lactamase bound tightly and specifically to IgG-Sepharose and could be stored for at least 4 weeks without dissociation. The rate of penicillin G hydrolysis by the beta-lactamase domain of the immobilized hybrid protein was found to depend on the amount of IgG covalently coupled to the support. For all IgG loads, higher specific activities and lower Km values relative to covalently immobilized beta-lactamase were obtained. Adsorption of the hybrid protein on the support resulted in increased stability to thermal deactivation. These results indicate that bifunctional hybrid proteins can be useful for the affinity immobilization of enzymes.
Chromatography, Affinity↗
Expression, purification, and immobilization of a protein A-beta-lactamase hybrid protein.
Explore the source record for details and available documents.
DNA, Recombinant↗
In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT.
The Escherichia coli outer membrane protease OmpT (protease VII) has been shown to degrade several proteins in vitro, but its function in vivo is uncertain. We demonstrate that OmpT participates in the degradation of a fusion protein secreted into the periplasmic space. A strain with mutations in degP (K.L. Strauch and J. Beckwith, Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988) and ompT exhibits a cumulative decrease in protein degradation and should be useful for the expression of proteolytically sensitive secreted proteins.
Bacterial Proteins↗
A study of serum free medium culture on the cloning efficiency of human bone marrow myeloid and erythroid progenitors.
Thirteen different combinations of serum-free media were tested to assess their suitability to replace serum containing medium for in vitro culture of human hemopoietic progenitors (CFU-GM and BFU-E). Bone marrow samples from patients with and without hematological diseases were tested. All tested media supported the growth of CFU-GM and BFU-E colonies, however our results have shown that the cloning efficiency of all commercially available serum-free media tested was lower (mean 18% and 12% of controls for CFU-GM and BFU-E respectively) and the colony size was smaller than those in serum-containing medium. In the serum-free cultures, there was no linear relationship between the colony numbers and cell concentration plated. Depletion of T-lymphocytes and monocytes did not improve the cloning efficiency of the serum-free medium culture. Furthermore, the addition of high concentration of insulin, transferrin and other supplements to the serum-free media did not improve the cloning efficiency. These results have indicated that the currently available commercial serum-free media do not provide optimal requirements for hemopoietic progenitor cell cultures and that other factors contained in serum are essential for their optimal growth.
Blood Physiological Phenomena↗
A human complement-fixing monoclonal anti-human lymphocyte antibody of rat origin.
MARCH 1E11 is an IgM monoclonal anti-human lymphocyte antibody of rat origin with the capacity to utilise both human and rabbit complement. The antibody reacts with all thymocytes and with all peripheral blood T and B lymphocytes. The treatment of human bone marrow or human peripheral blood mononuclear cells (PBMC) with MARCH 1E11 and either pooled human serum or autologous serum as a complement source resulted in cytolysis of greater than 99% of OKT3-positive lymphocytes. Under these conditions, progenitor cell recovery (colony-forming unit (CFU-c), burst forming unit--erythroid (BFU-e) and colony-forming unit--mixed (CFU-mix)) was greater than 90% of that of untreated cells. The response of treated marrow or PBMC to phytohaemagglutinin stimulation and in mixed leucocyte reactions demonstrated a reduction in thymidine incorporation to values similar to those obtained for unstimulated cells. The antibody does not cause modulation of the cell surface antigen and does not react significantly with non-lymphoid tissues. This monoclonal antibody may be useful for in vitro elimination of T lymphocytes from allogeneic bone marrow used for transplantation. The antibody may also be useful for treatment protocols requiring lymphoid depletion or immunosuppression as in organ transplantation.
Animals↗
Localization of inclusion bodies in Escherichia coli overproducing beta-lactamase or alkaline phosphatase.
High-level synthesis of the periplasmic protein beta-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. beta-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature beta-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.
Alkaline Phosphatase↗