ABSTRACT
BACKGROUND: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli. RESULTS: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface-anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l-1 could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique. CONCLUSIONS: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.
Subject(s)
Adhesins, Escherichia coli/metabolism , Bacterial Outer Membrane Proteins/genetics , Batch Cell Culture Techniques/methods , Escherichia coli/genetics , Gene Expression , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/genetics , Adhesins, Escherichia coli/genetics , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Culture Media/metabolism , Escherichia coli/growth & development , Escherichia coli/metabolism , Protein Engineering , Protein Sorting Signals , Protein Structure, Tertiary , Staphylococcal Protein A/metabolismABSTRACT
BACKGROUND: The production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes. RESULTS: A set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities. CONCLUSIONS: The use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.
Subject(s)
Acetic Acid/metabolism , Escherichia coli/metabolism , Glucose/metabolism , Membrane Proteins/biosynthesis , Escherichia coli/genetics , Escherichia coli/growth & development , Glucose/pharmacology , Membrane Proteins/genetics , MutationABSTRACT
The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h(-1) where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.
Subject(s)
Biotechnology/methods , Carboxylic Ester Hydrolases/metabolism , Escherichia coli/cytology , Escherichia coli/metabolism , Periplasm/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Carboxylic Ester Hydrolases/genetics , Cell Membrane/metabolism , Culture Media , Escherichia coli/growth & development , Immunoglobulin Fab Fragments/metabolism , Molecular Sequence Data , Recombinant Proteins , Time FactorsABSTRACT
In the present investigation, we have utilized the availability of UCP1(-/-) mice to examine a wide range of previously proposed lipid activators of Uncoupling Protein 1 (UCP1) in its native environment, i.e. in the brown-fat cells. A non-metabolizable fatty acid analogue, beta,beta cent-methyl-substituted hexadecane alpha,omega-dicarboxylic acid (Medica-16) is a potent UCP1 (re)activator in brown-fat cells, despite its bipolar structure. All-trans-retinoic acid activates UCP1 within cells, whereas beta-carotene only does so after metabolism. The UCP1-dependent effects of fatty acids are positively correlated with their chain length. Medium-chain fatty acids are potent UCP1 activators in cells, despite their lack of protonophoric properties in mitochondrial membranes. Thus, neither the ability to be metabolized nor an innate uncoupling/protonophoric ability is a necessary property of UCP1 activators within brown-fat cells.
Subject(s)
Adipose Tissue, Brown/metabolism , Fatty Acids/metabolism , Ion Channels/metabolism , Mitochondrial Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , Fatty Acids/pharmacology , Guanosine Diphosphate/metabolism , Kinetics , Mice , Mitochondria/metabolism , Oxygen Consumption/drug effects , Uncoupling Protein 1ABSTRACT
A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h(-1), respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.
Subject(s)
Escherichia coli/genetics , Genetic Engineering , Acetic Acid/metabolism , Culture Media , Escherichia coli/growth & development , Escherichia coli/metabolism , Glucose/metabolism , Oxygen ConsumptionABSTRACT
Mice with a dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutation (P465L) unexpectedly had normal amounts of adipose tissue. Here, we investigate the adipose tissue of the PPARgamma P465L mouse in detail. Microscopic analysis of interscapular adipose tissue of P465L PPARgamma mice revealed brown adipocytes with larger unilocular lipid droplets, indicative of reduced thermogenic capacity. Under conditions of cold exposure, the brown adipose tissue of the PPARgamma P465L mice was less active, a fact reflected in decreased uncoupling protein 1 levels. Analysis of the white adipocytes confirmed their normal cytoarchitecture and development, yet classical white adipose depots of the P465L PPARgamma mice had a striking reduction in brown adipocyte recruitment, a finding supported by reduced expression of UCP1 in the perigonadal adipose depot. Taken together, these data suggest that whole animal impairment of PPARgamma alters the cellular composition of the adipose organ to a more "white" adipose phenotype. Physiologically, this impairment in brown adipocyte recruitment is associated with decreased nonshivering thermogenic capacity after cold acclimation as revealed by norepinephrine responsiveness. Our results indicate that maintenance of oxidative brown-like adipose tissue is more dependent on PPARgamma function for development than white adipose tissue, an observation that may be relevant when considering PPARgamma-dependent strategies for the treatment of obesity.
Subject(s)
Adipocytes, Brown/physiology , PPAR gamma/genetics , PPAR gamma/physiology , Thermogenesis/genetics , Acclimatization/genetics , Acclimatization/physiology , Adipocytes, Brown/cytology , Adipose Tissue, Brown/anatomy & histology , Animals , Cell Count , Cold Temperature , Female , Gonads/cytology , Ion Channels/metabolism , Male , Mice , Mice, Transgenic , Mitochondrial Proteins/metabolism , Mutation, Missense , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Thermogenesis/physiology , Trans-Activators/metabolism , Transcription Factors , Uncoupling Protein 1ABSTRACT
During the recruitment process of brown adipose tissue, the mRNA level of the fatty acyl chain elongase Elovl3 is elevated more than 200-fold in cold-stressed mice. We have obtained Elovl3-ablated mice and report here that, although cold-acclimated Elovl3-ablated mice experienced an increased heat loss due to impaired skin barrier, they were unable to hyperrecruit their brown adipose tissue. Instead, they used muscle shivering in order to maintain body temperature. Lack of Elovl3 resulted in a transient decrease in the capacity to elongate saturated fatty acyl-CoAs into very long chain fatty acids, concomitantly with the occurrence of reduced levels of arachidic acid (C20:0) and behenic acid (C22:0) in brown adipose tissue during the initial cold stress. This effect on very long chain fatty acid synthesis could be illustrated as a decrease in the condensation activity of the elongation enzyme. In addition, warm-acclimated Elovl3-ablated mice showed diminished ability to accumulate fat and reduced metabolic capacity within the brown fat cells. This points to ELOVL3 as an important regulator of endogenous synthesis of saturated very long chain fatty acids and triglyceride formation in brown adipose tissue during the early phase of the tissue recruitment.
Subject(s)
Adipose Tissue/metabolism , Membrane Proteins/physiology , Acetyltransferases , Adipose Tissue, Brown/metabolism , Animals , Body Temperature , Body Weight , Chromatography, Gas , Cold Temperature , Crosses, Genetic , Eicosanoic Acids/chemistry , Electromyography , Fatty Acid Elongases , Fatty Acids/chemistry , Fatty Acids/metabolism , Female , Lipids/chemistry , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/metabolism , Oxygen Consumption , RNA/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Triglycerides/metabolismABSTRACT
We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.
Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Mitochondria/enzymology , Superoxide Dismutase/genetics , Uncoupling Agents/metabolism , Animals , Humans , Ion Channels , Mice , Mice, Transgenic , Mitochondrial Membranes/physiology , Mitochondrial Proteins , Muscles/enzymology , Oxidation-Reduction , Permeability , Superoxide Dismutase/biosynthesis , Superoxides/metabolism , Uncoupling Protein 1 , Uncoupling Protein 3ABSTRACT
Elevated circulating fatty acid concentration is a hallmark of insulin resistance and is at least in part attributed to the action of adipose tissue-derived tumor necrosis factor-alpha (TNF-alpha) on lipolysis. Cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) belongs to a family of proapoptotic proteins that has five known members in humans and mice. The action of CIDEA is unknown, but CIDEA-null mice are resistant to obesity and diabetes. We investigated CIDEA in adipose tissue of obese and lean humans and mice. The mRNA was expressed in white human fat cells and in brown mouse adipocytes. The adipose mRNA expression of CIDEA in mice was not influenced by obesity. However, CIDEA expression was decreased twofold in obese humans and normalized after weight reduction. Low adipose CIDEA expression was associated with several features of the metabolic syndrome. Human adipocyte depletion of CIDEA by RNA interference stimulated lipolysis and increased TNF-alpha secretion by a posttranscriptional effect. Conversely, TNF-alpha treatment decreased adipocyte CIDEA expression via the mitogen-activated protein kinase c-Jun NH(2)-terminal kinase. We propose an important and human-specific role for CIDEA in lipolysis regulation and metabolic complications of obesity, which is at least in part mediated by cross-talk between CIDEA and TNF-alpha.
Subject(s)
Adipocytes/physiology , Lipolysis/physiology , Obesity/physiopathology , Proteins/physiology , Adult , Animals , Apoptosis Regulatory Proteins , Cell Death , Cells, Cultured , Down-Regulation , Female , Gene Expression , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase 4 , Male , Mice , Mice, Inbred Strains , Middle Aged , Mitogen-Activated Protein Kinase Kinases/metabolism , Species Specificity , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.