Methods of Preparation of Recombinant Cytokines: IV. Renaturation of Recombinant Human Interleukin-3

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for renaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 × 10⁷ U/mg.     

Purification and renaturation of recombinant human interleukin-2.

Recombinant human interleukin-2 (IL-2) expressed as Escherichia coli was isolated as insoluble aggregates of protein (inclusion bodies) after cell breakage. IL-2 and contaminants were dissolved in 6 M-guanidinium chloride/10 mM-dithiothreitol, pH 8.5, and further purified in reduced and denatured form by gel-permeation chromatography in the same solvent. Renaturation was effected by dilution and autoxidation; IL-2 of native specific activity was isolated at over 95% purity by reversed-phase h.p.l.c.; an additional peak of reduced protein was also observed. Most losses of native IL-2 occurred on refolding, probably because of an aggregation process; concentrations around 1 microgram/ml were necessary to achieve 30% recovery. It was essential to maintain the denatured protein in reduced form before renaturation and autoxidation, which was most efficient at pH 8.5 with 1.5 microM-CuSO4. A procedure based on these observations has been used to prepare IL-2 on the 50 micrograms scale.     

Characterization of recombinant antibodies for cancer therapy by infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy was used to study the structure of the recombinant antibodies 1E10, anti-CD20 and hR3, which are used as anti-cancer therapeutic drugs. We tested their sensitivity against different conditions and treatments such as pH, temperature, freeze-thaw cycles and drying, which are relevant for the practical usefulness of the drugs. All antibodies were stable against moderate temperature increases (up to 50 °C) and pH changes (range 5–9). 1E10 was sensitive to extreme pH values (pH 3 and 12), whereas hR3 was most sensitive to temperature (at and above 60 °C). We did not observe any significant changes upon freeze-thaw and drying treatments. The secondary structure content of all three antibodies was estimated to be similar to that of IgG with ～64% β-sheet, 0% α-helix and ～36% other structure.     

Structural and functional properties of Escherichia coli-derived nucleoplasmin. A comparative study of recombinant and natural proteins.

Fourier transform infrared spectroscopy, circular dichroism and prediction techniques have been used to investigate the conformational properties of nucleoplasmin isolated from oocytes and eggs of Xenopus. laevis and overexpressed in Escherichia coli. A simple and fast method allows purification of recombinant nucleoplasmin free of truncated and/or aggregated forms, and therefore provides a suitable sample to carry out the structural and functional comparison between these proteins. The secondary structure of the three proteins estimated from both spectroscopic techniques was very similar, and was found to be 31--33% loops, 27--34% beta structure, 22--26% turns and 9-14% alpha helix. Prediction studies, in good agreement with experimental data, also suggest that beta structure is the major regular conformation, and that loops and turns are the most abundant conformational features within the secondary structure of nucleoplasmin. Furthermore, the spectroscopic characterization of a truncated version of the protein, lacking 80 residues at the C-terminus, and the prediction data indicate that the secondary structure elements of the protein are segregated into two regions. The N-terminal fragment (comprising residues 1--120) which holds all the putative beta strands, and the solvent-exposed C-terminal region, that is suggested to be enriched in turn and loop structures. The phosphate/protein monomer molar ratios, obtained from chemical analysis and mass spectrometry, are 0, 3 and 7--10 for recombinant, oocyte and egg nucleoplasmin, respectively. Phosphorylation does not significantly affect the secondary structure of the protein, but clearly modulates its ability to decondense sperm nuclei and to remove basic proteins from DNA.     

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Nucleic acid can catalyze the conversion of α‐helical cellular prion protein to β‐sheet rich Proteinase K resistant prion protein oligomers and amyloid polymers in vitro and in solution. Because unfolding of a protein molecule from its ordered α‐helical structure is considered to be a necessary step for the structural conversion to its β‐sheet rich isoform, we have studied the unfolding of the α‐helical globular 121–231 fragment of mouse recombinant prion protein in the presence of different nucleic acids at neutral and acid pH. Nucleic acids, either single or double stranded, do not have any significant effect on the secondary structure of the protein fragment at neutral pH; however the protein secondary structure is modified by the nucleic acids at pH 5. Nucleic acids do not show any significant effect on the temperature induced unfolding of the globular prion protein domain at neutral pH which, however, undergoes a gross conformational change at pH 5 as evidenced from the lowering of the midpoint of thermal denaturation temperatures, Tm, of the protein. The extent of Tm decrease shows a dependence on the nature of nucleic acid. The interaction of nucleic acid with the nonpolar groups exposed from the protein interior at pH 5 probably contributes substantially to the unfolding process of the protein.     

Crystal structure of recombinant human interleukin-4.

The crystal structure of recombinant human interleukin-4 (rhuIL-4) was initially determined at 3.5-A resolution by multiple isomorphous replacement techniques and subsequently refined to a resolution of 2.35 A by simulated annealing. The final crystallographic R-factor, based on all data in the range 6.0-2.35 A (7470 reflections), is 0.232. Bond lengths and bond angles in the molecule have root mean square deviations from ideal values of 0.016 A and 2.4 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhuIL-4 is a four alpha-helix bundle, which composes approximately 58% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within these connections is a two-stranded antiparallel beta-sheet. Both the tertiary and secondary structures of rhuIL-4 are similar to those of human granulocyte-macrophage colony-stimulating factor. Critical regions for receptor binding are proposed.     

Studies of the structure of bacteriophage lambda cro protein in solution. Analysis of the circular dichroism data

The secondary and tertiary structures of bacteriophage cro protein were studied by circular dichroism. The pH dependence of this structure was investigated: cro protein is stable over pH 4.5-10.5. At these pH-values cro protein contains approximately 35% alpha-helix, approximately 20% antiparallel beta-structure and approximately 15% beta-turn, while the remaining part of the protein molecule is in the irregular state. The secondary and tertiary structures of the protein are modified abruptly at more acid and more alkaline pH-values. The curves characterizing the secondary and tertiary structures of the protein are symbatic. The effect of Gu-HCl on the secondary and tertiary structures of cro protein at 22 degrees C and pH 7.2 was studied also. The conformational transition occurs within 0.6-1.9 M Gu-HCl. The changes in the secondary and tertiary structures of the protein have a symbatic character. Thermal denaturation of cro protein was examined. A possible mechanism of the protein denaturation is discussed.     

Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.     

Conformation and self-association of human recombinant transforming growth factor-beta3 in aqueous solutions

The transforming growth factors-beta (TGF-beta) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-beta isoforms TGF-beta3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-beta3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-beta3. In addition, the aggregation of TGF-beta3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-beta3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH approximately 3.9, a monomer right arrow over left arrow dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8. 5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-beta3 dissolved in solution and aggregated TGF-beta3. Aggregated TGF-beta3 showed a higher beta-sheet content and lower beta-turn and random coil contributions compared with monomeric TGF-beta3. Both the solution structure and the aggregate structure of TGF-beta3 were different from the crystal structure. This was in contrast to TGF-beta2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-beta3 conformation in solution in the range of 2.3 < pH < 11. 0 was reversible. Aggregation of TGF-beta3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, cNaCl = 164 mM) TGF-beta3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations.     

Assembly of the full-length recombinant mouse prion protein I. Formation of soluble oligomers

The conversion of a monomeric alpha-helix-rich isoform to multimeric beta-sheet-rich isoforms is a prominent feature of the conversion between PrP(C) and PrP(SC). We mimicked this process in vitro by exposing an unglycosylated recombinant form of the full-length mouse prion protein ((Mo)PrP(23-231)) to an acidic pH, at 37 degrees C, and we monitored the kinetics of conformational change and assembly. In these conditions, monomeric (Mo)PrP(23-231) converts slowly to two ensembles of soluble oligomers that are separated by size exclusion chromatography. The larger oligomers (I) are unstable, and their formation involves almost no change in secondary structure content. The smaller oligomers (II) form stable spherical or annular particles containing between 8 and 15 monomers as determined by multi-angle laser light scattering (MALLS). Their formation is concomitant with the main, thought limited, change in the secondary structure content (10%) seen by Fourier Transform Infrared (FTIR) spectroscopy. Even if these oligomers conserve a large part of the secondary structure of monomeric PrP, they exhibit amyloid features with the appearance of intermolecular beta-structure as revealed by the appearance of an IR band below 1620 cm(-1).     

Circular dichroism of reduced and oxidized recombinant human epidermal growth factor.

To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r-HuEGF) we studied the structure of reduced and oxidized r-HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r-HuEGF in 10 mM sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of alpha-helix, beta-sheet, and unordered structure. The addition of the alpha-helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of alpha-helix, at the apparent expense of beta-sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r-HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding.     

A new approach for determining the stability of recombinant human epidermal growth factor by thermal Fourier transform infrared (FTIR) microspectroscopy

Based on Fourier transform infrared (FTIR) microspectroscopy, the conformation of rhEGF under the influence of pH, heat treatment, chaotropic salts, concentration of salt and protein structure perturbants was studied. The FTIR spectrum of rhEGF showed that major secondary structures from amide I bands composed of 40.6% beta-sheets, 25.0% reverse turns, 16.5% random coils, 13.0% loops and 4.9% side-chain structures. At extreme pH conditions (pH < 4 and pH > 8), there were changes in intensity of the bands attributed to loop (1658 cm(-1)) and random coil structures, and these bands shifted to lower wavenumbers, indicating changes in protein conformation. Thermal denaturation of rhEGF occurred at 40-76 degrees C and the formation of intermolecular beta-aggregates was revealed by the FTIR spectra. Thermal-irreversible property of rhEGF after second-heating treatment suggested that rhEGF has a poor thermal stability. While investigating the stability of rhEGF in the presence of chaotropic salts, anions induced protein unfolding of rhEGF more significantly than cations. The optimal stabilizing effect was found at the 2 M NaCl added to rhEGF, and expressed the structure of rhEGF more stable on the many components. The bands of loop structure (1654 cm(-1)), beta-sheet (1638 cm(-1)) and intermolecular antiparallel beta-aggregation formation (1694, 1619 and 1612 cm(-1)) seem to be "marked" to be more sensitive in determining environmental changes of rhEGF for FTIR microspectroscopy.     

Intermediates in denaturation of a small globular protein, recombinant human stefin B.

Guanidinium HCl (GdmHCl), pH, and heat denaturation of the recombinant human stefin B, a low molecular weight protein inhibitor of cysteine proteinases, has been followed by circular dichroism. From the noncoincidence of the transitions in the near and far UV, the existence of stable intermediate states possessing few persistent tertiary interactions but most of the native-like secondary structure, was inferred. These intermediate states exist at equilibrium under various conditions, namely, state G at 1.7 M GdmHCl (pH 8, 25 degrees C), state A at pH 4 (0.6 M GdmHCl, 25 degrees C) and state T above 68 degrees C. By size exclusion chromatography, their apparent compactness was determined. The intermediate states A, T, and G were compact and are therefore classified as "molten globule" states.     

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.     

Structure and pH-induced alterations of recombinant and natural spider silk proteins in solution

The spinning process of spiders can modulate the mechanical properties of their silk fibers. It is therefore of primary importance to understand what are the key elements of the spider spinning process to develop efficient industrial spinning processes. We have exhaustively investigated the native conformation of major ampullate silk (MaS) proteins by comparing the content of the major ampullate gland of Nephila clavipes, solubilized MaS (SolMaS) fibers and the recombinant proteins rMaSpI and rMaSpII using (1) H solution NMR spectroscopy. The results indicate that the protein secondary structure is basically identical for the recombinant protein rMaSpI, SolMaS proteins, and the proteins in the dope, and corresponds to a disordered protein rich in 3(1) -helices. The data also show that glycine proton chemical shifts of rMaSpI and SolMaS are affected by pH, but that this change is not due to a modification of the secondary structure. Using a combination of NMR and dynamic light scattering, we have found that the spectral alteration of glycine is concomitant to a modification of the hydrodynamical diameter of recombinant and solubilized MaS. This led us to suggest new potential roles for the pH acidification in the spinning process of MaS proteins.     

Crystallization of purified recombinant human interleukin-1 beta

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.     

Isolation of recombinant interleukin-3 produced by E. coli]

A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.     

Conformational stability of human erythrocyte transglutaminase. Patterns of thermal unfolding at acid and alkaline pH.

Tissue-type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0-9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition-state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH, we noted that transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-terminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C-terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N-terminal and C-terminal regions interact with each other in determining the structure of the native protein.     

Mimicking lipid-binding-induced conformational changes in the human apolipoprotein E N-terminal receptor binding domain effects of low pH and propanol.

We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1-191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1-191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross-linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu-C, as is the case for apo E3(1-191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n-propanol increases the amount of calculated alpha-helical structure (42-65%) and alters the tertiary structure of the protein although not as much as when apo E3(1-191) is incorporated into lipid bicelles. In the absence of n-propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n-propanol at pH 4.5 increases the Stokes radius of apo E3(1-191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0-4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu-C. As assessed by far and near UV circular dichroism, the addition of 30% n-propanol at pH 4.5, in contrast to pH 7, markedly increases the alpha-helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1-191) into lipid bicelles. The results suggest that a combination of n-propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids.