Quality and authenticity of heterologous proteins synthesized in yeast.

Yeast, especially Saccharomyces cerevisiae and Pichia pastoris, are major hosts employed in the expression of authentic heterologous proteins of high quality in the biopharmaceutical, industrial and academic environments. There has been recent progress in characterizing and controlling the factors involved in determining authenticity.

Genetic stability of protein expression systems in yeast.

For the expression of recombinant proteins in yeast, genetic stability is monitored using a combination of standard microbiology and nucleic acid testing procedures. Process consistency during the cell amplification and product expression phases of fermentation are also reliable indicators of stability. The potential for instability arising from point mutation, gene conversion or recombination has been shown to occur at a low frequency and does not generally affect protein product quality.

Transport of substrates and metabolites and their effect on cell metabolism (in butyric-acid and methylotrophic fermentations).

The two components, delta pH and delta psi, of the membrane protonmotive force (delta p) effect and are affected by the transport of many substrates and metabolites. Because the integrity (or restoration) of the delta p requires the expenditure of metabolic energy, such transport processes affect the overall cell bioenergetics. However, the transport or high concentrations of certain substrates and metabolites can have more serious effects on cell metabolism because they partially or completely abolish either or both the delta pH and delta psi. If the cells cannot eventually restore the collapsed component(s) of the delta p, complete growth inhibition and cell death become inevitable. In the butanol/acetone fermentation of Clostridium acetobutylicum, the transport and the presence of key metabolites (acetic and butyric acids, and butanol) have serious and some necessary effects on the delta p. Acetic and butyric acids act as uncouplers of the delta pH, thereby reducing the internal pH. Using other acid uncouplers (such as acetoacetate, which is metabolized by the cells, or FCCP, which is not metabolized by the cells), we found that a lower pHo combined with the metabolic-energy drain of the uncoupling effect and high internal acid concentrations are implicated in the mechanism(s) of solventogenesis. Thus, the production or presence (or both) of the two acids (acetic and butyric) is beneficial to the initiation of solvent production. The transport mechanisms of CH3OH, CH2O, and HCOOH in obligate CH3OH utilizers (methylotrophs) were also discussed in detail. We showed that CH3OH is actively transported by the cells at the expense of metabolic energy and that its transport significantly affects the dynamics of continuous bioreactors. The accumulation of CH2O was found to be driven by the membrane delta p. Finally, formate was accumulated by the delta pH according to the general transport mechanism of short-chain fatty acids. The inhibition of growth by formate was explained by its uncoupling effect on the cells. Growth inhibition by CH3OH appeared to be related to the severe reduction of the membrane delta pH and cell pHi by relatively low CH3OH concentrations.

Direct measurement of carbon substrate oxidation and incorporation patterns in RuMP-type methylotrophs: Chemostatic cultures of Methylomonas L3.

A technique using C-14 isotope tracers to probe the branching of carbon flow in methylotrophic bacteria has been devised and applied to continuous steady-state cultures. Methylomonas L3, a strain which utilizes the KDPG/TA variant of the ribulose monophosphate cycle for carbon fixation, was employed in the experimental studies. The actual in vivo rates of substrate-carbon incorporation into biomass, both direct and via CO(2), and of the two carbon oxidation schemes were determined in three different steady-state cultures. The results show that the carbon substrate is oxidized predominantly via formate (the linear oxidation scheme), and that the cyclic scheme of oxidation is minimally, if at all, utilized. The carbon incorporation and oxidation patterns appear to vary considerably with the dilution rate and the inoculum history. The experimental accuracy of the new technique is discussed in detail.

Turnover of brain postsynaptic densities after selective deafferentation: detection by means of an antibody to antigen PSD-95.

Antibodies to antigen PSD-95, a neuronal protein present only in postsynaptic densities (PSDs), have been used to follow immunohistochemically the loss and recovery of PSDs after selective deafferentation of both the hippocampal formation and the lateral septal nucleus of the rat. Three days after unilateral entorhinal ablation, densitometry of brain tissue sections stained by the peroxidase-antiperoxidase (PAP) method showed a decrease of about 25% in the outer 2/3 of the ipsilateral dentate gyrus molecular layer (ML), whereas staining in the inner 1/3 of the same ML layer actually increased about 16%. The intensely staining inner 1/3 of the deafferented ML expanded over time so that by 30 days postlesion the expansion had reached 60-70% of the ML. In the lateral septal nucleus, unilateral fimbria transection did not change either the pattern of anti-PSD PAP staining or that of [125I]protein A binding as revealed by autoradiography or by microdissection of the lateral septal nuclei to determine bound radioactivity by gamma-counting. Bilateral intraventricular injection of kainic acid to destroy area CA3 of the ipsilateral hippocampus caused very little loss of anti-PSD stain in the dentate gyrus ML, but decreased by 45% and 34% the intensity of stain in stratum radiatum and stratum oriens of the hippocampus, respectively. However, bilateral injections of doses of kainate high enough to destroy areas CA3, CA4 and part of CA1, caused 53% loss of stain in the inner 1/3 of the dentate gyrus ML. Recovery from the PSD loss caused by kainate in area CA1 was slow, only 67-81% of control by 90 days post-lesion. The immunohistochemical results in dentate gyrus and septum corresponded closely with quantitative data obtained by electron microscopy. Therefore, the response of PSDs to the loss of their presynaptic counterpart appears to depend on the overall extent of deafferentation of the neuron, the zone of the dendritic tree where deafferentation occurs and, perhaps, other specific features of the target cells.

Isolation, morphology, and protein and glycoprotein composition of synaptic junctional fractions from the brain of lower vertebrates: antigen PSD-95 as a junctional marker.

Synaptic junction (SJ) fractions were isolated from the brains of the gray shark, bonito, common frog, several reptiles, and common chicken and compared to those prepared from mammalian brain. All SJ preparations, as judged by electron microscopic analysis, were at least 85% pure, consisting primarily of postsynaptic densities (PSDs) with or without an overlying plasma membrane and, to a lesser extent, of complete synaptic junctions. Complete junctions were less abundant in preparations from lower vertebrates. The electrophoretic pattern of SJs from different vertebrate species showed considerable conservation of the major protein bands. The most abundant were fibrous proteins, especially tubulins, actin, and the PSD-specific polypeptide with a molecular weight of 52,000 (PSD-52). Glycoproteins capable of binding concanavalin A were present in SJs from all vertebrates; their apparent molecular weight and relative abundance were characteristic of each animal order examined, showing more similarities in species more closely related phylogenetically. Finally, a protein (antigen PSD-95) previously shown to be located specifically in the postsynaptic densities of the mammalian brain was present in all species. The binding of antibody specific to this protein decreased with descending phylogenetic order from mammals to shark. Nonetheless, PSD-95 was present in all vertebrate species and appeared to be a general specific marker for PSDs.

Postsynaptic density antigens: preparation and characterization of an antiserum against postsynaptic densities.

Long-term immunization of rabbits with postsynaptic densities (PSD) from bovine brain produced an antiserum specific for PSD as judged by binding to subcellular fractions and immunohistochemical location at the light and electron microscope levels. (a) The major antigens of bovine PSD preparations were three polypeptides of molecular weight 95,000 (PSD-95), 82,000 (PSD-82), and 72,000 (PSD-72), respectively. Antigen PSD-95, also present in mouse and rat PSDs was virtually absent from cytoplasm, myelin, mitochondria, and microsomes from rodent or bovine brain. Antigens PSD-82 and PSD-72 were present in all subcellular fractions from bovine brain, especially in mitochondria, but were almost absent from rodent brain. The antiserum also contained low-affinity antibodies against tubulin. (b)Immunohistochemical studies were performed in mouse and rat brain, where antigen PSD-95 accounted for 90 percent of the antiserum binding after adsorption with purified brain tubulin. At the light microscope level, antibody binding was observed only in those regions of the brain where synapses are known to be present. No reaction was observed in myelinated tracts, in the neuronal cytoplasm, or in nonneuronal cells. Strong reactivity was observed in the molecular layer of the dentate gyrus, stratum oriens and stratum radiatum of the hippocampus, and the molecular layer of the cerebellum. Experimental lesions, such as ablation of the rat entorhinal cortex or intraventricular injection of kainic acid, which led to a major loss of PSD in well- defined areas of the hippocampal formation, caused a correlative decrease in immunoreactivity in these areas. Abnormal patterns of immunohistochemical staining correlated with abnormal synaptic patterns in the cerebella of reeler and staggerer mouse mutants. (c) At the electron microscopic level, immunoreactivity was detectable only in PSD. The antibody did not bind to myelin, mitochondria or plasma membranes. (d) The results indicate that antigen PSD-95 is located predominantly or exclusively in PSD and can be used as a marker during subcellular fractionation. Other potential uses include the study of synaptogenesis, and the detection of changes in synapse number after experimental perturbations of the nervous system.

Optimal concentration of iodonitrotetrazolium for the isolation of junctional fractions from rat brain.

The yield and purity of synaptic plasma membranes (SPM) and synaptic junctions (SJ) from rat brain has been examined as a function of the concentration of rho-iodonitrotetrazolium (INT)--succinate used during their preparation. An INT concentration of 1 mg/g brain tissue (wet weight) was sufficient to obtain SPM and SJ of purity comparable to that obtained using 4--6 times that concentration of dye (1--3). At this lower level of INT the yield of SPM increased by about 100%, whereas mitochondrial contamination remained at 10--13% of the total SPM protein. At concentrations of INT below 0.5 mg/g brain tissue (wet weight) the contamination of SPM by mitochondria increased rapidly. At very low concentrations of INT (0.13 mg/g tissue) the contaminating protein of mitochondrial origin was 40--50% of the total protein in the SPM fraction. Examination by gel electrophoresis of SPM, SJ, and mitochondrial fractions with different degrees of cross-contamination allowed the assignment of marker polypeptides for mitochondrial, junctional, and nonjunctional plasma membranes. Under the conditions used to prepare SJ, a variable amount of particulate material floated over 1.0 M sucrose. It consisted of SJ and many membrane vesicles and had a protein composition similar to that of SJ contaminated by extrajunctional membrane proteins. An analogous fraction arose during in the preparation of postsynaptic densities.