Improved pertussis toxin production by Bordetella pertussis through adjusting the growth medium's ionic composition.

Ionic composition and total ionic concentration of the growth medium were important factors in limiting productivities in aerated reactors used for the production of pertussis toxin and other antigens by Bordetella pertussis. Salt concentration has opposing effects on cell growth of wild-type B. pertussis and specific toxin formation. Sodium ion concentrations below 140 mM correlated with a precipitous decline in specific yields of pertussis toxin, an otherwise growth-associated product. High salt concentrations in the medium resulted in lower final cell concentrations but did not affect initial growth rates. A new medium is proposed that allows a 60 to 70% increase in both cell and toxin yields by replacing the sodium chloride in the 'cyclodextrin liquid' (CL) medium with additional monosodium glutamate which provides both the sodium and the carbon and energy source.

Thermal unfolding of bacteriophage T4 short tail fibers.

The short tail fibers of bacteriophage T4 are composed of a homotrimer of the product of gene 12 (P12) with a molecular weight of 165,000. P12 is capable of reconstituting defective phage particles lacking gene 12. After heating to 75 degrees C, P12 was able to retain 90% of its ability to reconstitute T412- particles. When heated above 75 degrees C, P12 was no longer capable of fully reconstituting defective phage particles. By 95 degrees C, the reconstitution efficiency of the P12 preparation was reduced by 4 orders of magnitude. Thermal unfolding was also monitored by heating the protein in the presence of SDS to freeze partially unfolded states; by protease hydrolysis; and by intrinsic fluorescence changes. Exposure to SDS had little effect for temperatures up to 55 degrees C, but by 65 degrees C, the reconstitution efficiency of P12 treated with 0.01% SDS dropped to less than 1% of the original titer. Thermolysin digestion of P12 heated to various temperatures showed that treated P12 started to inactivate before 45 degrees C and inactivation was essentially complete by 55 degrees C. Intrinsic fluorescence data of heated P12 indicated that the protein begins to unfold by 45 degrees C and exhibits distinct peak shifts at 60 degrees C and above 80 degrees C. We conclude that, in the absence of SDS or proteases, P12 heated to 75 degrees C can refold back to an active conformation. Trimeric P12 undergoes some irreversible denaturation between 75 and 85 degrees C, and heating between 85 and 95 degrees C results in dissociation of P12 into monomers.

Oxidative renaturation of hen egg-white lysozyme. Folding vs aggregation.

Since the inception of recombinant DNA technology, different strategies have been developed in the isolation, renaturation, and native disulfide bond formation of proteins produced as insoluble inclusion bodies in Escherichia coli. One of the major challenges in optimizing renaturation processes is to prevent the formation of off-pathway inactive and aggregated species. On the basis of a simplified kinetic model describing the competition between folding and aggregation, it was possible to analyze the effects of denaturant and thiol/disulfide concentrations on this competition. Although higher guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields, the folding rate was negatively affected, indicating an optimum range of GdmCl for optimum renaturation rates and yields. Similarly, higher total glutathione concentrations resulted in higher yields but decreased rates, also indicating an optimum total glutathione concentration for optimum renaturation rates and yields (6-16 mM), with an optimum ratio of reduced to oxidized glutathione between 1 and 3. To characterize the nature of aggregates, aggregation experiments were performed under different oxidizing/reducing conditions. It is shown that hydrophobic interactions between partially folded polypeptide chains are the major cause of aggregation. Aggregation is fast and aggregate concentration does not significantly increase beyond the first minute of renaturation. Under conditions which promote disulfide bonding, aggregate size, but not concentration, may increase due to disulfide bond formation, resulting in covalently bonded aggregates.

Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme.

The effect of typical contaminants in inclusion body preparations such as DNA, ribosomal RNA, phospholipids, lipopolysaccharides, and other proteins on renaturation rate and yield of hen egg white lysozyme was investigated. Separate experiments were conducted in which known amounts of individual contaminants were added to test their effect on renaturation kinetics. On the basis of a simplified model for the kinetic competition between folding and aggregation, it was found that none of the above contaminants had an effect on the rate of the folding reaction, but some of them significantly affected the rate of the aggregation reaction and, thus, the overall renaturation yield. While ribosomal RNA did not seem to affect the aggregation reaction, plasmid DNA and lipopolysaccharides increased the aggregation rate, resulting in a decrease of about 10% in the overall renaturation yield. Phospholipids were found to improve refolding yields by about 15% by decreasing the overall rate of the aggregation reaction without affecting the rate of the folding reaction. Proteinaceous contaminants which aggregate upon folding, such as beta-galactosidase and bovine serum albumin, were found to significantly decrease renaturation yields by promoting aggregation. This effect was strongly dependent on the concentration of the proteinaceous impurity. On the other hand, the presence of refolding ribonuclease A, which does not significantly aggregate upon folding under the conditions tested in this work, did not affect the renaturation kinetics of lysozyme, even at concentrations as high as 0.7 mg/mL.

Oxidative renaturation of hen egg-white lysozyme in polyethylene glycol-salt aqueous two-phase systems.

Aqueous two-phase systems have been widely used for the separation and concentration of proteins. In this work we investigated the possibility of using aqueous two-phase system for the renaturation of inclusion body proteins by studying the effect of polyethylene glycol (PEG)-salt systems on the oxidative renaturation of hen egg-white lysozyme (HEWL) with guanidinium chloride (GdmCl) present in the system. To accomplish phase separation at moderately low concentrations of polymer and salt, the total GdmCl concentration had to be kept low (<1 M). The unfolded protein exhibited very low solubility under these conditions. In an attempt to increase the solubility of the protein, temperatures of 40, 50, and 60 degrees C were investigated. The effect of PEG molecular weight was also addressed. Best renaturation yields were obtained when using PEG 3400 and working at 50 degrees C. However, the total protein concentration had to be kept at a low level of 0.2 mg/mL. Lowering the total GdmCl concentration in the system resulted in increased aggregation.

Oxidative renaturation of lysozyme at high concentrations.

Newly synthesized cloned gene proteins expressed in bacteria frequently accumulate in insoluble aggregates or inclusion bodies. Active protein can be recovered by solubilization of inclusion bodies followed by renaturation of the solubilized (unfolded) protein. The recovery of active protein is highly dependent on the renaturation conditions chosen. The renaturation process is generally conducted at low protein concentrations (0.01-0.2 mg/mL) to avoid aggregation. We have investigated the potential of successfully refolding reduced and denatured hen egg white lysozyme at high concentrations (1 and 5 mg/mL). By varying the composition of the renaturation media, optimum conditions which kinetically favor proper folding over inactivation were found. Solubilizing agents such as guanidinium chloride (GdmCl) and folding aids such as L-arginine present in low concentrations during refolding effectively enhanced renaturation yields by suppressing aggregation resulting in reactivation yields as high as 95%. Quantitatively the kinetic competition between lysozyme folding and aggregation can be described using first-order kinetics for the renaturation reaction and third-order kinetics for the overall aggregation pathway. The rate constants for both reactions have been found to be strongly dependent on denaturant and thiol concentration. This strategy supercedes the necessity to reactivate proteins at low concentrations using large renaturation volumes. The marked increase in volumetric productivity makes this a viable option for recovering biologically active protein efficiently and in high yield in vitro from proteins produced as inclusion bodies within microbial cells.

Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor.

Recombinant human macrophage colony-stimulating factor (rhM-CSF), a homodimeric, disulfide bonded protein, was expressed in Escherichia coli in the form of inclusion bodies. Reduced and denatured rhM-CSF monomers were refolded in the presence of a thiol mixture (reduced and oxidized glutathione) and a low concentration of denaturing agent (urea or guanidinium chloride). Refolding was monitored by nonreducing gel electrophoresis and recovery of bioactivity. The effects of denaturant type and concentration, protein concentration, concentration of thiol/disulfide reagents, temperature, and presence of impurities on the kinetics of rhM-CSF renaturation were investigated. Low denaturant concentrations (<0.5 M urea) and high protein concentrations (>0.4 mg/ml) in the refolding mixture resulted in increased formation of aggregates, although aggregation was never significant even when refolding was carried out at room temperature. Higher protein concentration resulted in higher rates but did not lead to increased yields, due to the formation of unwanted aggregates. Experiments conducted at room temperature resulted in slightly higher rates than those conducted at 4 degrees C. Although the initial renaturation rate for solubilized inclusion body protein without purification was higher than that of the reversed-phase purified reduced denatured rhM-CSF, the final renaturation yield was much higher for the purified material. A maximum refolding yield of 95% was obtained for the purified material at the following refolding conditions: 0.5 M urea, 50 mM Tris, 1.25 mM DTT, 2 mM GSH, 2 mM GSSG, 22 degrees C, pH 8, [protein] = 0.13 mg/ml.

Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans.

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures.