A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of alpha-chymotrypsin denatured with 8 M urea and 100 mM dithiothreitol at pH 8.2. The complete activity could be regained within 10 min during refolding. Both native and refolded enzymes showed emission of intrinsic fluorescence with lambda(max) of 342 nm. Gel electrophoresis showed that the presence of Eudragit S-100 led to dissociation of multimers followed by the appearance of a band at the monomer position. The unfolding (by 8 M urea) and folding (assisted by the polymer) also led to complete renaturation of alpha-chymotrypsin initially denatured by 90% dioxane. The implications of the data in recovery of enzyme activity from inclusion bodies and the interesting possibility in the in vivo context of reversing protein aggregation in amyloid-based diseases have been discussed.

Alkaline conditions are generally preferred for sanitization of chromatography media by cleaning-in-place (CIP) protocols in industrial biopharmaceutical processes. The use of such rigorous conditions places stringent demands on the stability of ligands intended for use in affinity chromatography. Here, we describe efforts to meet these requirements for a divalent proteinaceous human serum albumin (HSA) binding ligand, denoted ABD*dimer. The ABD*dimer ligand was constructed by genetic head-to-tail linkage of two copies of the ABD* moiety, which is a monovalent and alkali-stabilized variant of one of the serum albumin-binding motifs of streptococcal protein G. Dimerization was performed to investigate whether a higher HSA-binding capacity could be obtained by ligand multimerization. We also investigated the influence on alkaline stability and HSA-binding capacity of three variants (VDANS, VDADS and GGGSG) of the inter-domain linker. Biosensor binding studies showed that divalent ligands coupled using non-directed chemistry demonstrate an increased molar HSA-binding capacity compared with monovalent ligands. In contrast, equal molar binding capacities were observed for both types of ligands when using directed ligand coupling chemistry involving the introduction and recruitment of a unique C-terminal cysteine residue. Significantly higher molar binding capacities were also detected when using the directed coupling chemistry. These results were confirmed in affinity chromatography binding capacity experiments, using resins containing thiol-coupled ligands. Interestingly, column sanitization studies involving exposure to 0.1 M NaOH solution (pH 13) showed that of all the tested constructs, including the monovalent ligand, the divalent ligand construct containing the VDADS linker sequence was the most stable, retaining 95% of its binding capacity after 7 h of alkaline treatment.

It was shown recently that proline-beta-naphthylamidase from pig liver resembles the gamma-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and V(max)/K(m) values from 12 to 1000 l min(-1) x 10(-3) using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190-210 kDa. All enzymes exhibit the highest activity in the pH range 8-9 and between 60 and 70 degrees C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-beta-naphthylamidase activity than rPLE.

The influence of different factors acting on Escherichia coli periplasmic expression of recombinant human growth hormone (hGH) in shake flask cultures has been investigated. Bacterial vectors containing the phage lambdaP(L) promoter, which is temperature activated, were utilized. Four different signal peptides were compared: DsbA, npr, STII and one derived from the natural hGH signal peptide, this last used as a reference. Other factors such as medium composition, optimized induction and expression conditions, and different bacterial strains were also studied. The determination of hGH, carried out directly in osmotic shock fluids, was based on an isocratic reversed-phase high-performance liquid chromatography method, which allows direct, rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space immediately after or even during fermentation. The level of hGH production increased 2.5-fold compared with the reference vector, reaching a level of 3.9 +/- 0.63 micro g/ml/A(600) (n = 6; coefficient of variation = 16.2%). The expression level was affected by the signal peptide and by the induction conditions, being more effective when activation started in the early logarithmic phase which, however, exhibited remarkably different optical density (OD) according to medium composition. Our results thus indicate that 6 h activation at 40-42 degrees C, starting with an OD (A(600)) of approximately 3 in a very rich medium, were conditions capable of providing the maximum secretion level for a vector utilizing the DsbA signal sequence and E.coli W3110 or RB791 as host cells.

Recently, we designed a short alpha-helical fibril-forming peptide (alphaFFP) that can form alpha-helical nanofibrils at acid pH. The non-physiological conditions of the fibril formation hamper biomedical application of alphaFFP. It was hypothesized that electrostatic repulsion between glutamic acid residues present at positions (g) of the alphaFFP coiled-coil sequence prevent the fibrillogenesis at neutral pH, while their protonation below pH 5.5 triggers axial growth of the fibril. To test this hypothesis, we synthesized alphaFFPs where all glutamic acid residues were substituted by glutamines or serines. The electron microscopy study confirmed that the modified alphaFFPs form nanofibrils in a wider range of pH (2.5-11). Circular dichroism spectroscopy, sedimentation, diffusion and differential scanning calorimetry showed that the fibrils are alpha-helical and have elongated and highly stable cooperative tertiary structures. This work leads to a better understanding of interactions that control the fibrillogenesis of the alphaFFPs and opens opportunities for their biomedical application.

The ribosome display system is a very effective and powerful tool for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. The system depends on the stability of ribosome-mRNA complexes that have been formed as a result of the removal of a stop codon. To assess the general applicability of the system, we examined the stability of ribosome-mRNA complexes in the presence and absence of a stop codon, as well as in the presence and the absence of an additional interaction between the translated peptide and its mRNA within the ribosome-mRNA complex. The additional interaction that we exploited was the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (Cv). The MSp dimer and Cv were placed, respectively, at the N-terminal end of a nascent protein, translated in vitro, and at the 5' end of the protein's mRNA, and consequently further stabilize the ribosome-mRNA complex. To our surprise, we were able to select proteins even in the presence of a stop codon. Moreover, as we had anticipated, the interaction between the MSp dimer and Cv enhanced the stability of the ribosome-mRNA complex, suggesting that this kind of interaction might be useful in the design of an efficient ribosome display selection strategy. Indeed, the yield of the mRNAs of interest after selection was increased upon the introduction of the interaction between the MSp dimer and Cv.

Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours. The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, epsilon-NH(2) groups in lysine side chains. In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues. The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position. One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF. EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents. Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes. These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.

We previously reported a method, designated functional salvage screen (FSS), to generate protein lineages with new sequence spaces through the functional or structural salvage of a defective protein by employing green fluorescent protein (GFP) as a model protein. Here, in an attempt to mimic a step in the natural evolution process of proteins, the functionally salvaged mutant GFP-I5 with new sequence space, but showing low fluorescence intensity and stability, was selected and fine-tuned by directed evolution. During a course of functional tuning, GFP-I5 was found to evolve rapidly, recovering the spectral traits to those of the parent GFPuv. The mutant 3E4 from the third round of directed evolution possessed four substitutions; three (F64L, E111V and K166Q) were at the original GFP gene and the other (K8N) at the inserted segment. The fluorescence intensity of 3E4 was approximately 28-fold stronger than GFP-I5, and other spectral properties were retained. Biochemical and biophysical investigations suggested that the fine-tuning by directed evolution led the salvaged variant GFP-I5 to a functionally favorable structure, resulting in recovery of stability and fluorescence. Site-directed mutagenesis of the mutated amino acid residues in both GFPuv and GFP-I5 revealed that each amino acid residue has a different effect on the fluorescence intensity, which implies that 3E4 adopted a new evolutionary path with respect to fluorescence characteristics compared with the parent GFPuv. Directed evolution in conjunction with FSS is expected to be used for generating protein lineages with new fitness landscapes.

The binding affinity and specificity of recombinant antibodies can be modified by site-directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine-tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen-binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.

Stimulation of T-cells by IL-2 has been exploited for treatment of metastatic renal carcinoma and melanoma. However, a narrow therapeutic window delimited by negligible stimulation of T-cells at low picomolar concentrations and undesirable stimulation of NK cells at nanomolar concentrations hampers IL-2-based therapies. We hypothesized that increasing the affinity of IL-2 for IL-2Ralpha may create a class of IL-2 mutants with increased biological potency as compared with wild-type IL-2. Towards this end, we have screened libraries of mutated IL-2 displayed on the surface of yeast and isolated mutants with a 15-30-fold improved affinity for the IL-2Ralpha subunit. These mutants do not exhibit appreciably altered bioactivity at 0.5-5 pM in steady-state bioassays, concentrations well below the IL-2Ralpha equilibrium binding constant for both the mutant and wild-type IL-2. A mutant was serendipitously identified that exhibited somewhat improved potency, perhaps via altered endocytic trafficking mechanisms described previously.

ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl-moiety (AMP approximately) of ATP to sulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP(i)). Subsequently, APS kinase mediates transfer of the gamma-phosphoryl group of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and ADP. The recently determined crystal structure of yeast ATP sulfurylase suggests that its C-terminal domain is structurally quite independent from the other domains, and not essential for catalytic activity. It seems, however, to dictate the oligomerization state of the protein. Here we show that truncation of this domain results in a monomeric enzyme with slightly enhanced catalytic efficiency. Structural alignment of the C-terminal domain indicated that it is extremely similar in its fold to APS kinase although not catalytically competent. While carrying out these structural and functional studies a surface groove was noted. Careful inspection and modeling revealed that the groove is sufficiently deep and wide, as well as properly positioned, to act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme.

The role of the long loop connecting beta-strands F5 and F6 (21 amino acids, Pro302-Leu-Asp-Arg-Thr-Lys-Ser-Pro-Leu-Ser-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu322) present in Rhodotorula gracilis d-amino acid oxidase (RgDAAO) was investigated by site-directed mutagenesis. This loop was proposed to play an important role in the 'head-to-tail' monomer-monomer interaction of this dimeric flavoenzyme: in particular, by means of electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3" of the other monomer. We produced a mutant of RgDAAO (namely, DAAO-DeltaLOOP2), in which only minor structural perturbations were introduced (only five amino acids were deleted; new sequence of the betaF5-betaF6 loop is Pro302-Leu-Asp-Arg-Thr-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu317), and the charge of the betaF5-betaF6 loop not modified. The DeltaLOOP2 mutant is monomeric, has a weaker binding with the FAD cofactor, a decrease of the kinetic efficiency, and slight modifications in its spectral properties. The short version of the loop does not allow a correct monomer-monomer interaction, and its presence in the monomeric DAAO is a destabilizing structural element since the DeltaLOOP2 mutant is highly susceptible to proteolysis. These results, confirming the role of this loop in the subunits interaction and thus in stabilization of the sole dimeric form of RgDAAO, put forward the evidence that even a short deletion of the loop generates a consistent variation of the enzyme structure-function properties.

An intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F. F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions. In the presence of SDS, wild-type human CDA dissociates into enzymatically inactive monomers without intermediate forms via a non-cooperative transition. Extensive dialysis or dilution of the inactivated monomers restores completely the activity. Circular dichroism measurements show that the secondary/tertiary structure organization of each subunit is unaffected by the SDS concentration, while the mutation Phe/Trp causes weakening in quaternary structure. The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavours dissociation of the tetramer into subunits in the wild-type CDA, but not in mutant enzyme F137W/W113F. The absence of tyrosine fluorescence and the much higher quantum yield of the double mutant protein spectrum suggest the occurrence of an energy transfer effect between the protein subunits. This assumption is confirmed by the crystallographic studies on B.subtilis in which it is shown that three different subunits concur with the formation of each of the four active sites and that F125, homologous to the human CDA F137, is located at the interface between two different subunits contributing to the formation of active site.

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) >> His39Cys > His39Ser.

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.