‘Chillum’ jar,a better technique for screening of rhizobia under summer conditions

Summary In the present investigations ‘Chillum’ jar assembly was found to provide more favourable environmental conditions for rhizobia to nodulate leguminous plants particularly under summer conditions than the usual Leonard jar assembly. When thirty pigeon pea rhizobia isolates were tested for their nodulation efficiency in both Leonard jars as well as ‘Chillum’ jars, it was noticed that there was no nodulation in any of the isolates under Leonard jars whereas all isolates were nodulating well under ‘Chillum’ jars conditions. This was probably due to lowering of temperature in ‘Chillum’ jar caused by rapid evaporation from the outer surface of ‘Chillum’ jar assembly. The maximum temperature recorded in ‘Chillum’ jar was 34°C whereas in Leonard jars it was 46.5°C.     

Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR?×?anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.     

A circularly permuted β-lactamase as a novel reporter for evaluation of protein cyclization efficiency

Split inteins have been used as a versatile tool in protein engineering to mediate efficient in vivo and in vitro trans-splicing of a protein. The trans-splicing ability of split inteins was also applied to the in vivo cyclization of a protein. However, cyclization efficiency is dependent upon the type of split inteins employed and the conditions under which cyclization occur. In this study, a novel reporter system that easily measures the cyclization efficiency of split inteins was developed. For this purpose TEM-1 beta-lactamase was divided into two fragments (24 approximately 215 and 216 approximately 286 amino acids) and circularly permuted. The circularly permuted beta-lactamase expressed in Escherichia coli showed little beta-lactamase activity, most likely due to the structural modification of the protein. However, when the circularly permuted beta-lactamase was cyclized by the Synechocystis sp. PCC6803 DnaB split mini-intein, beta-lactamase activity both in vitro and in vivo was recovered. These results suggest that the novel reporter system can be exploited to develop new inteins with high efficiency of in vivo protein cyclization.     

Quantitative analysis of trace gases of breath during exercise using the new SIFT–MS technique

Abstract

We show how the concentration of the breath gases ammonia, acetone, and isoprene vary with time during exercise using the new selected ion flow tube mass spectrometry (SIFT–MS) technique. The expired breath concentrations of ammonia, acetone and isoprene were observed within the range of 50–500, 100–1400 and 5–400 ppb, respectively. Increasing acetone levels were observed for most subjects during the exercise period. However, isoprene levels decreased with time during exercise. Older subjects showed higher levels of isoprene compared with younger subjects. The ammonia time profile with exercise showed both decreasing and increasing patterns for different subjects.     

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c ₂ interacting with cytochrome bc ₁ complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein–protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.     

Bacterial production and refolding from inclusion bodies of a “Weak” toxin, a disulfide rich protein

The gene for the “weak” toxin of Naja kaouthia venom was expressed in Escherichia coli. “Weak” toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of “weak” toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia “weak” toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain “weak” toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of ¹²⁵I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α1₂β1_γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC₅₀ = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH₄C₁ cell line also showed the presence of activity close to that of the natural toxin (IC₅₀ 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain ¹⁵N-labeled analog of the neurotoxin.     

Tracking a protein following dissociation from a protein–protein complex using a split SNAP-tag system

Protein–protein interactions (PPIs) are important for various biological processes in living cells. Several methods have been developed for the visualization of PPIs in vivo; however, these methods are unsuitable for visualization of post-PPI events such as dissociation and translocation. In this study, we applied a split SNAP-tag system for the visualization of post-PPI events. This method enabled tracking of the protein following dissociation from the protein–protein complex. Thus, the split SNAP-tag system should prove to be a useful tool for visualization of post-PPI events.     

Optimisation of OPLS–UA force-field parameters for protein systems using protein data bank

We have previously proposed a method for refining force-field parameters of protein systems, which consists of minimising the summation of the square of the force acting on each atom in the proteins with the structures from the protein data bank (PDB). The results showed that the modified force-field parameters for all-atom model gave structures more consistent with the experimental implications than the original force fields. In this work, we applied this method and a new method to the OPLS–UA force field. In the new method, we perform a minimisation of the average of the root-mean-square deviation of various protein structures from the native structure. We selected some torsion-energy parameters for this optimisation, and 100 molecules from the PDB were used. The results imply that the new force-field parameters gave structures of two peptides more consistent with the experimental implications for the secondary structure-forming tendencies than the original OPLS–UA force field.     

Quantitative structure–activity relationship analysis using deep learning based on a novel molecular image input technique

Quantitative structure–activity relationship (QSAR) analysis uses structural, quantum chemical, and physicochemical features calculated from molecular geometry as explanatory variables predicting physiological activity. Recently, deep learning based on advanced artificial neural networks has demonstrated excellent performance in the discipline of QSAR research. While it has properties of feature representation learning that directly calculate feature values from molecular structure, the use of this potential function is limited in QSAR modeling. The present study applied this function of feature representation learning to QSAR analysis by incorporating 360° images of molecular conformations into deep learning. Accordingly, I successfully constructed a highly versatile identification model for chemical compounds that induce mitochondrial membrane potential disruption with the external validation area under the receiver operating characteristic curve of ≥0.9.     

High-efficiency recovery of target cells using improved yeast display system for detection of protein–protein interactions

We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein–protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fc part of human immunoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.     

Helix-mediated protein–protein interactions as targets for intervention using foldamers

Protein–protein interactions (PPIs) play a central role in virtually all biological processes and have been the focus of intense investigation from structural molecular biology to cell biology for the majority of the last two decades and, more recently, are emerging as important targets for pharmaceutical intervention. A common motif found at the interface of PPIs is the α-helix, suggesting that, in the same way as the “lock and key” model has evolved for competitive inhibition of enzymes, it should be possible to elaborate “rule-based” approaches for inhibition of helix-mediated PPIs. This review will describe the biological function and structural features of a series of representative helix-mediated PPIs and discuss approaches that are being developed to target these interactions with small molecules that employ non-natural amino acids.     

Feasibility of protein spray coating using a fluid-bed Würster processor

This article documents a feasibility study on coating fine powders with protein solutions using a Würster spray coater (GPCG-1 from Glatt Air Techniques, Ramsey, NJ). Spray coating was based on a fluid-bed process where fluidized microcarriers were coated inside the Würster column and dried in the fluidization chamber. Recombinant human deoxyribonuclease (rhDNase) was used as the model protein. Lactose powders of two different size ranges, 53-125 and 125-250 mum, were used as the model microcarrier. The amount of protein applied was varied to obtain coatings of varying thickness. The extent of rhDNase loading determined experimentally was found to be consistent with the theoretical value and was also confirmed visually by scanning electron microscopy. The coating showed a strong integrity after being subjected to mechanical force. However, the protein suffered serious aggregation during coating, most likely due to the thermal stress of the process. Aggregation was significantly reduced when rhDNase was formulated with calcium ions, consistent with the observation that Ca(2+) thermally stabilized the protein (as determined by scanning microcalorimetry) in aqueous solution. Thus, our study demonstrates that spray coating, particularly when used in conjunction with rational stabilization strategies, is a feasible alternative to other methods of preparing dried pharmaceutical proteins. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 560-566, 1997.     

Simultaneous assay of muscarinic and β-adrenergic receptors using a double isotope technique

Muscarinic receptor and β-adrenergic receptor binding were measured simultaneously in a membrane fraction of bovine tracheal smooth muscle using [³H]-L-quinuclidinyl benzilate and [¹²⁵I]-(?)iodocyanopindolol. The binding characteristics, affinity and receptor density, obtained in the double receptor assay and in the control experiments were the same within experimental error. Moreover, there appears to be neither a significant influence of an excess of d,l-propranolol on [³H]-L-quinuclidinyl benzilate binding nor a significant influence of an excess of l-quinuclidinyl benzilate on [¹²⁵I]-(?)iodocyanopindolol binding. The method is advantageous where both receptors have to be assayed and where limited amounts of biological material, like in biopsy specimen, are available.     

The use of Quantitative Agarose Gel Electrophoresis for rapid analysis of the integrity of protein–DNA complexes

Recent biochemical studies evaluated the affinity of histones to DNA in the context of nucleosome core particle (NCP). These have indicated a concentration-dependence for nucleosome stability. However, when studying chromatin the preferred templates are nucleosome arrays (NA) and not the NCP. Biochemical methods are poorly suited for structural analysis of chromatin. To overcome that technical hindrance, and investigate the effect of concentration on stability of the histone–DNA interactions, we have applied the multigel Quantitative Agarose Gel Electrophoresis (QAGE) method to in vitro-assembled nucleosomal arrays. The results demonstrated the method to be extremely valuable for the evaluation of the effect of low concentration on NA. However, QAGE is a fairly time-demanding and complex method. To maximize the efficiency of use of this technology, we devised a protocol that allowed for multiple sets of templates to be analyzed simultaneously. Briefly, samples can be loaded at regular intervals and analyzed individually for their molecular composition. The technique presented in this study describes the calibration steps and proof of concept necessary to validate the use of multiple loading of multigel to evaluate the composition of nucleosomal arrays as a function of concentration.     

Synthesis and evaluation of a library of 2,5-bisdiamino-benzoquinone derivatives as probes to modulate protein–protein interactions in prions

A small library combining two different benzoquinone cores with seven (l) amino acid methyl esters (alanine, Nω-nitro-arginine, Nε-BOC-lysine, isoleucine, methionine, phenylalanine and tryptophan) was prepared and tested for prion replication inhibition in ScGT1 cells. The most potent hit, 6a, displayed an EC₅₀ value of 0.87 μM, which is very close to that of quinacrine (0.4 μM).     

Construction and evaluation of a novel bifunctional phenylalanine–formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration

Phenylalanine dehydrogenase (PheDH) plays an important role in enzymatic synthesis of l-phenylalanine for aspartame (sweetener) and detection of phenylketonuria (PKU), suggesting that it is important to obtain a PheDH with excellent characteristics. Gene fusion of PheDH and formate dehydrogenase (FDH) was constructed to form bifunctional multi-enzymes for bioconversion of l-phenylalanine coupled with coenzyme regeneration. Comparing with the PheDH monomer from Microbacterium sp., the bifunctional PheDH–FDH showed noteworthy stability under weakly acidic and alkaline conditions (pH 6.5–9.0). The bifunctional enzyme can produce 153.9 mM l-phenylalanine with remarkable performance of enantiomers choice by enzymatic conversion with high molecular conversion rate (99.87 %) in catalyzing phenylpyruvic acid to l-phenylalanine being 1.50-fold higher than that of the separate expression system. The results indicated the potential application of the PheDH and PheDH–FDH with coenzyme regeneration for phenylpyruvic acid analysis and l-phenylalanine biosynthesis in medical diagnosis and pharmaceutical field.     

Quantitative comparison of dynamic physiological feeding profiles for recombinant protein production with Pichia pastoris

Pichia pastoris is widely used for the production of recombinant proteins in industrial biotechnology. In general, industrial production processes describe fed-batch processes based on the specific growth rate. Recently, we introduced the specific substrate uptake rate (q _s) as a novel parameter to design fed-batch strategies for P. pastoris. We showed that a dynamic feeding strategy where the feed was adjusted in steps to the maximum specific substrate uptake rate was superior to more traditional strategies in terms of specific productivity. In the present study, we compare three different dynamic feeding strategies based on q _s for a recombinant P. pastoris strain with respect to cell physiology, methanol accumulation, productivity and product quality. By comparing (A) a feeding profile at constant high q _s, (B) a periodically adjusted feeding profile for a stepwise q _s ramp, and (C) a feeding profile at linear increasing q _s, we evaluated potential effects of the mode of feeding. Although a dynamic feeding strategy with stepwise increases of q _s to q _{s max} resulted in the highest specific productivity, a feeding profile where the feeding rate was stepwise increased to a constant high q _s value was superior in terms of the amount of active enzyme produced and in the amount of accumulated methanol. Furthermore, this feeding strategy could be run automatically by integrating an online calculator tool, thus rendering manual interventions by the operator unnecessary.     

Design of new reaction conditions for characterization of a mutant thermophilic α-l-fucosidase

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.