Frank Hoffmann

The small heat-shock proteins IbpA and IbpB reduce the stress load of recombinant Escherichia coli and delay degradation of inclusion bodies

BackgroundThe permanently impaired protein folding during recombinant protein production resembles the stress encountered at extreme temperatures, under which condition the putative holding chaperones, IbpA/IbpB, play an important role. We evaluated the impact of ibpAB deletion or overexpression on stress responses and the inclusion body metabolism during production of yeast α-glucosidase in Escherichia coli.ResultsDeletion of ibpAB, which is innocuous under physiological conditions, impaired culture growth during α-glucosidase production. At higher temperatures, accumulation of stress proteins including disaggregation chaperones (DnaK and ClpB) and components of the RNA degradosome, enolase and PNP, was intensified. Overexpression of ibpAB, conversely, suppressed the heat-shock response under these conditions. Inclusion bodies of α-glucosidase started to disaggregate after arrest of protein synthesis in a ClpB and DnaK dependent manner, followed by degradation or reactivation. IbpA/IbpB decelerated disaggregation and degradation at higher temperatures, but did hardly influence the disaggregation kinetics at 15°C. Overexpression of ibpAB concomitant to production at 42°C increased the yield of α-glucosidase activity during reactivation.ConclusionsIbpA/IbpB attenuate the accumulation of stress proteins, and – at high temperatures – save disaggregated proteins from degradation, at the cost, however, of delayed removal of aggregates. Without ibpAB, inclusion body removal is faster, but cells encounter more intense stress and growth impairment. IbpA/IbpB thus exert a major function in cell protection during stressful situations.

Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris.

A single‐chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol‐inducible AOX1 promoter. In high‐cell‐density cultivation on defined medium, methanol‐limited and methanol‐saturated conditions were compared. After batch and fed‐batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single‐step downstream processing from the culture supernatant by His‐affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.

Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway.

The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by lon-deficient Bl21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon.

Optimized Production of Active α‐Glucosidase by Recombinant Escherichia coli. Evaluation of Processes Using in Vivo Reactivation from Inclusion Bodies

During fast production in recombinant Escherichia coli, the enzyme alpha-glucosidase from Saccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusion bodies are reactivated inside the cell upon temperature downshift. This in vivo reactivation was most efficient on complex medium with inclusion body production at 42 degrees C and reactivation at 30 degrees C, yielding volumetric activities 85% higher than those of extended isothermal production at low temperature. On defined medium, however, in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specific growth rate independent of the temperature. Here, high growth rates fostered inclusion body formation even at low temperature. Intermediate growth rates permitted accumulation of active alpha-glucosidase without affecting the total amount of alpha-glucosidase. Low growth rates yielded similar activities and additionally prevented inclusion body formation. Moreover, high growth rates during production forestalled subsequent in vivo reactivation. Accumulation of activity after temperature reduction was possible with intermediate growth rates. The best time for temperature shift was concomitant to induction. Thus, in fed-batch culture, isothermal production at 30 degrees C and with a set growth rate of 0.12 h(-)(1) controlled by feeding was most efficient for production of active alpha-glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.

Single-chain antibody fragment production in Pichia pastoris: Benefits of prolonged pre-induction glycerol feeding

Secretory production of a single‐chain antibody fragment (scFv) by recombinant Pichia pastoris using the methanol inducible AOX1 promoter is limited biochemically by retarded secretion, and economically by the high demand for pure oxygen. To address the problem, the adaptation phase with growth‐limiting feeding of glycerol before the production phase was optimized. In a standard procedure with a short glycerol‐feeding phase before induction, scFv accumulated in the supernatant only after 15 h. Conversely, scFv started to appear immediately in the medium upon methanol induction when the glycerol‐feeding phase was extended to 18 h. Interestingly, despite a significantly lower cell density in the cultivation with extended glycerol feeding, the same amount of functional product of 300 mg/L was obtained about 30 h after the start of glycerol feeding with both methods. mRNA analysis revealed that the higher and faster production of the product was related to longer lasting induction of the scFv mRNA. Additional effects of a better adaptation of the secretion machinery may be suggested by higher expression of unfolded protein response‐related genes KAR2 and PDI. A clear benefit of the longer glycerol‐feeding phase was a 75% reduction of the consumption of both pure oxygen and methanol, and a significantly lower cell density, which would be beneficial for down‐stream purification of the product.

Unsymmetrically disubstituted mesogens with a phenyl ring containing lateral branch. Crystal and molecular structure of smectogenic 4-nitrobenzyl 2-(3-chloro-4-n-octyloxybenzoyloxy)-5-(4-n-octyloxybenzoyloxy)benzoate

Abstract The synthesis of unsymmetrically disubstituted mesogens bearing a cyclic unit within the bulky lateral branch is described. The three-ring basic mesogenic unit of the laterally 4-nitrobenzyloxy-carbonyl substituted molecules has been additionally provided with chlorine atoms in one or two positions, thus influencing the stability of the smectic A phases. The crystal and molecular structure of the smectogenic 4-nitrobenzyl 2-(3-chloro-4-n-octyloxybenzoyloxy)-5-(4-n-octyloxybenzoyloxy)benzoate (NCOOB) has been determined by X-ray analysis. NCOOB crystallizes in the triclinic space group P1 with 4 molecules per unit cell and the following lattice parameters: a = 13.201(2) A, b = 16.473(3) A, c = 19.746(3) A, α = 96.11(2)°, β = 95.34(2)°, γ = 99.92(1)°, V = 4178.4(1) A3. The phenylene bis(n-octyloxybenzoate) mesogenic basic fragment of the NCOOB molecule has a non-planar, but optimally stretched shape, with a perfect all-trans-conformation of the alkyloxy chains and the lateral benzene ring-containin...

Synthesis and Chemical Constitution of Diphenoxyphosphoryl Derivatives and Phosphonium Salts as Coupling Reagents for Peptide Segment Condensation

The reactions of diphenoxyphosphoryl chloride ((PhO) 2 P(O)Cl) and different chlorophosphonium salts ([R 3 PCl]X, R = (CH 3 ) 2 N, pyrrolidine, X = PF 6 m , BF 4 m ), respectively, with 7-aza-1-hydroxybenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), hydroximinomalonitrile (HOxDCO), and ethyl hydroximinocyanoacetate (HOxO) are described. The structures of the new compounds, which are useful coupling reagents for epimerization-free peptide segment condensation, are discussed on the basis of their 1 H, 13 C, 31 P NMR, and IR spectra. The reactions of (PhO) 2 P(O)Cl lead to mixtures of O - and N -phosphorylated isomers of varying ratios. Contrary, reactions of chlorophosphonium salts yield exclusively one isomer.

Crystal and Molecular Structure of the Laterally Branched Nematogenic Compound 2-n-Nonyl-1,4-phenylene Bis (4-n-octyloxybenzoate)

Abstract The crystal and molecular structure of the nematogenic compound 2-n-nonyl-1,4-phenylene bis(4-n-octyloxybenzoate) (NPOB) has been determined at – 100°C and at room temperature by X-ray analysis. NPOB crystallizes in space group P1 with two molecules in a unit cell of dimensions (values of the room temperature investigation are given in brackets) a = 9.878(2) [10.165(4)], b = 12.700(3) [12.863(5)], c = 17.830(4) [17.940(6)] A, α = 71.83(2) [69.65(2)], β = 84.58(2) [85.23(2)], γ = 76.11(2) [75.43(2)]°. The structure was solved by direct methods and refined to an R value of 0.056 [0.108]. The most striking features of the molecular structure are the all-trans-conformation of the laterally branched nonyl group and the nearly parallel alignment of all three alkyl substituents. The crystal structure is characterized by a parallel arrangement and an interlocked packing of the molecules.

Crystal and Molecular Structure of a Mesogenic Compound with a Large Lateral Benzene Ring-containing Substituent

Abstract The crystal and molecular structure of the nematic compound 4-nitrobenzyl 2,5-bis (4-ethyloxybenzoyloxy)-benzoate (NEBB) has been determined at −100°C and at room temperature by X-ray analysis. NEBB crystallizes in space group P 1 with two molecules in a unit cell of dimensions (values of the room temperature investigation are given in brackets) a = 9.547 (3) [9.643 (2)], b = 12.294 (4) [12.452 (3)], c = 13.536 (5) [13.660 (3)] A, α = 68.67 (3) [68.34(1)], β= 75.15 (3) [74.40 (1)], γ = 70.98 (3) [69.65 (1)]°. The structure was solved by direct methods and refined on F 2 to R values of wR2 = 0.107 [0.109] and R1 = 0.043 [0.049], respectively, for 3347 [2422] observed unique reflections. The phenylene bis(benzoate) basic core of the NEBB molecule has a non-planar but optimally stretched shape and the lateral ring-containing substituent is oriented parallel to it. The molecules in the crystalline state are arranged perfectly parallel to each other with an anti-parallel orientation of the neighbours....

Novel Mesogenic Benzoic Acids with Large Branches I. Synthesis, Liquid Crystalline Properties and Crystal Structure Analyses of 3-(4-Subst.-benzyloxycarbonyl)-4-(4-n-octyloxybenzoyloxy) benzoic Acids

Abstract New benzoyloxybenzoic acids bearing both terminal and lateral groups have been synthesized. Substituted aryl rings are laterally attached to the basic mesogens by means of an odd-numbered spacer. Therefore, liquid crystalline behaviour can be observed at high temperatures. The crystal and molecular structure of 3-(4-cyano-benzyloxycarbonyl)-4-(4-n-octyloxybenzoyloxy)benzoic acid 3b has been determined by X-ray analysis. 3b crystallizes in space group P21/n with four molecules in a unit cell of the following dimensions: a = 15.682(2), b = 7.701(1), c = 23.107(3)A β = 97.69(1)°. The structure was solved by direct methods and refined to an R value of 0.057 for 3684 observed reflections. The molecules of 3b are associated to dimers with a shape unusual for mesogens exhibiting nematic and smectic phases. In the crystal the dimers are closely packed to ribbons which on their part are connected to lamellar sheets. The nitro-substituted homologue 3c was proved to be isostructural with 3b.