First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.     

Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用

在前期研究中,已发现人瘦素（leptin）在体外再折叠过程中会形成稳定的二聚体,但其二聚化机制尚不清楚. 本研究旨在分析瘦素二聚体的结构特性,并重点研究体外再折叠过程中瘦素二聚化的机制. 相较与瘦素单体,瘦素二聚体保留了约75％免疫活性及15％受体结合活性,同时显示出明显慢的天然电泳迁移率. 圆二色性分析显示,二聚体基本保留了单体α螺旋索结构特征. 还原性及非还原性凝胶电泳分析和自由巯基测定结果表明,瘦素二聚体是由一对分子间二硫键连接2个单体而成的.为了确定瘦素二聚化过程中起主导作用的分子间二硫键,利用PCR定点突变技术构建了C96S和C146S两个突变体瘦素. 通过分析C96S及C146S突变体瘦素的体外再折叠特性及过程,并与野生型瘦素相比较,揭示C96S瘦素的二聚体显示出与野生型瘦素二聚体相似的特性,而C146S瘦素不能形成结构稳定的二聚体. 以上研究结果表明,Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用.     

Design of highly stable functional GroEL minichaperones.

GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins. With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL(193-345). A sequence alignment of 130 members of the chaperonin 60 (Cpn60) family was used to design 37 single mutations. Two small-to-large mutations, A223T, A223V and one similar-size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol(-1) each. Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol(-1) more stable than wild-type protein. Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive. In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis. In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein.     

Recombinant Human Ribosomal Protein S16: Expression,Purification, Refolding,and Structural Stability

The cDNA of human ribosomal protein S16 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in E. coli cells and highly purified protein was isolated. A method for refolding the protein from inclusion bodies was optimized. The secondary structure content of the refolded protein was analyzed by CD spectroscopy. It was found that 21 +/- 4% of the amino acid sequence of the protein forms alpha-helices and 24 +/- 3% is in beta-strands. The protein structure stability was studied at various pH values and urea concentrations. The protein is quickly denatured at pH above 8.0, whereas increasing of urea concentration causes slow unfolding of the protein.     

A Cold Acclimation Protein with Refolding Activity on Frozen Denatured Enzymes

We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH₄)₂SO₄ precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100°C, cryopreservation at -20°C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12°C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82×10^-10, 4.35×10^-9, 8.98×10^-12, and 3.05×10^-11, respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.     

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

With advancements in high‐throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chemical denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample volumes 10‐50‐fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodynamic and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.     

Light waveguiding in bioinspired peptide nanostructures

Basic optical properties of bioinspired peptide nanostructures are deeply modified by thermally mediated refolding of peptide secondary structure from α‐helical to β‐sheet. This conformational transition is followed by the appearance in the β‐sheet structures of a wideband optical absorption and fluorescence in the visible region. We demonstrate that a new biophotonic effect of optical waveguiding recently observed in peptide/protein nanoensembles is a structure‐sensitive bimodal phenomenon. In the primary α‐helical structure input, light propagates via optical transmission window demonstrating conventional passive waveguiding, based on classical optics. In the β‐sheet structure, fluorescent (active) light waveguiding is revealed. The latter can be attributed to completely different physical mechanism of exciton‐polariton propagation, characterized by high effective refractive index, and can be observed in nanoscale fibers below diffraction limit. It has been shown that peptide material requirements for passive and active waveguiding are dissimilar. Original biocompatibility and biodegradability indicate high potential future applications of these bioinspired waveguiding materials in precise photobiomedicine towards advanced highly selective bioimaging, photon diagnostics, and optogenetics.     

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (C_H1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between V_H-C_H1 and V_L-C_L was improved by the C_H1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.     

Factors affecting protein refolding yields in a fed-batch and batch-refolding system

The refolding of recombinant protein from inclusion bodies expressed in Escherichia coli can present a process bottleneck. Yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. This article studies the effect of five factors upon the dilution refolding of protein in a twin impeller fed-batch system using refold buffer containing only the oxidized form of the redox reagent. Such a buffer is easier to prepare and more stable than a buffer containing both reduced and oxidized forms. The five factors chosen were: bulk impeller Reynolds number, mini-impeller Reynolds number, injection rate of denatured protein, redox ratio, and guanidine hydrochloride (GdHCl) concentration. A 2(5) factorial experiment was conducted at an industrially relevant protein concentration using lysozyme as the test system. The study identified that in the system used, the guanidine hydrochloride concentration, redox ratio, and injection rate were the most important factors in determining refolding yields. Two interactions were found to be important: redox ratio/guanidine hydrochloride concentration and guanidine hydrochloride concentration/injection rate. Conditions were also found at which high refolding yields could be achieved even with rapid injection and poor mixing efficiency. Therefore, a comparative assessment was carried out with minimal mixing in a simple batch-refolding mode of operation, which revealed different behavior to that of fed-batch. A graphical (windows of operation) analysis of the batch data suggested that optimal yields and productivity are obtained at high guanidine hydrochloride concentrations (1.2 M) and redox ratios of unity or greater.