Isolation and quantitation of isopentenyladenosine in an anise cell culture by single-ion monitoring,radioimmunoassay and bioassay

Using three different techniques, isopentenyladenosine was identified and quantitated in an anise cell line (Pimpinella anisum L.), growing on a medium without cytokinin. A method to quantitate cytokinins was developed which utilizes extraction of cytokinins in the presence of a deuterated reference compound and subsequent quantitation of the cytokinins by single-ion monitoring on a gas chromatography-mass spectrometry system. The results were compared with those obtained by radioimmunoassay as well as a bioassay. Quantitations by gas chromatography-mass spectrometry and radioimmunoassay correlated well, whereas the estimates by the bioassay gave considerably lower values.     

Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

三种禽病毒抗原的串联表达、纯化及活性鉴定

为实现多个基因在同一菌株中均一可溶性表达,简化基因工程亚单位多联多价疫苗中抗原生产的工艺步骤,本研究选用Ⅰ群4型禽腺病毒(FAdV-4) Fiber-2蛋白、鸡传染性法氏囊病病毒(IBDV) VP2蛋白和减蛋综合征病毒(EDSV)Fiber蛋白3种来自不同禽病毒的抗原为研究对象,利用原核表达系统,通过密码子优化、载体启动子改造和基因串联顺序优化,获得单一载体/多重转录单元的共表达重组质粒。将共表达重组质粒转化大肠杆菌BL21(DE3)菌株,进行3个基因的共表达。纯化后的蛋白进行Western blotting和蛋白活性检测。结果表明,目的基因经过密码子优化、载体启动子改造和基因串联顺序的优化后,获得均一可溶性共表达的3种蛋白,纯化后蛋白纯度大于80%,Western blotting分析和琼脂扩散试验表明串联表达的3种蛋白具有免疫反应性和抗原活性。文中通过目的基因密码子优化、表达载体启动子改造和基因串联等关键技术的突破,首次实现了3种不同禽病毒抗原的高效、均一、可溶性串联表达和纯化,为基因工程亚单位多联多价疫苗的研制奠定了基础。     

High-level expression and purification of an analgesic peptide from Buthus martensii Karch

BmK AngM1, a scorpion peptide isolated from Buthus martensii Karch was reported to exhibit potential analgesic effect. But the relative low content of this toxin in crude venom limits its further characterization. In this study, we constructed an expression vector and transformed into E.coli. The BmK AngM1 was expressed as a fusion protein in the soluble fraction and was purified by Nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. We purified 25 mg recombinant BmK AngM1 (rBmK AngM1) from 1 L bacterial culture. The molecular weight of rBmK AngM1 determined by ESI-MS was 7240.4 Da which was the expected size for correctly processed. Analgesic bioassay studies of rBmK AngM1 exhibited its potential analgesic effect comparable to that of the natural BmK AngM1 peptide.     

Evaluation of the properties of potassium ferrate used for water purification by luminescence bioassay

Characteristics of four natural water samples from urban and rural areas and the efficiency of a new purifying agent, potassium ferrate K₂FeO₄, were studied by bacterial luminescence bioassay for 30 minutes. It was revealed that two samples of water from the urban areas are toxic, while the other two samples (one from urban and one from rural environment) are nontoxic. Numerous data obtained on the increase in toxicity index with time allow reasonable conclusions to be made about the chemical nature of substances present in the test water samples. Toxic natural water samples were likely to contain heavy metals and were well purified using potassium ferrate, including via their adsorption. In nontoxic natural water samples, toxic complexes with organic compounds present in water could form at the addition of potassium ferrate. The obtained data call for further studying the properties of potassium ferrate complexes with organic compounds. Bacterial luminescence bioassay is a promising method for the rapid assessment of properties of various water sources (their integral toxicity and presumable chemical composition) and new reagents for their purification (effective concentrations, bactericidal properties, and mechanisms of interacting with heavy metals and organic substances in water).     

Preparative peptide purification by cation-exchange and reversed-phase perfusion chromatography.

Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.     

Synaptic inhibition in an isolated nerve cell

Following the preceding studies on the mechanisms of excitation in stretch receptor cells of crayfish, this investigation analyzes inhibitory activity in the synapses formed by two neurons. The cell body of the receptor neuron is located in the periphery and sends dendrites into a fine muscle strand. The dendrites receive innervation through an accessory nerve fiber which has now been established to be inhibitory. There exists a direct peripheral inhibitory control mechanism which can modulate the activity of the stretch receptor. The receptor cell which can be studied in isolation was stimulated by stretch deformation of its dendrites or by antidromic excitation and the effect of inhibitory impulses on its activity was analyzed. Recording was done mainly with intracellular leads inserted into the cell body. 1. Stimulation of the relatively slowly conducting inhibitory nerve fiber either decreases the afferent discharge rate or stops impulses altogether in stretched receptor cells. The inhibitory action is confined to the dendrites and acts on the generator mechanism which is set up by stretch deformation. By restricting depolarization of the dendrites above a certain level, inhibition prevents the generator potential from attaining the "firing level" of the cell. 2. The same inhibitory impulse may set up a postsynaptic polarization or a depolarization, depending on the resting potential level of the cell. The membrane potential at which the inhibitory synaptic potential reverses its polarity, the equilibrium level, may vary in different preparations. The inhibitory potentials increase as the resting potential is displaced in any direction from the inhibitory equilibrium. 3. The inhibitory potentials usually rise to a peak in about 2 msec. and decay in about 30 msec. After repetitive inhibitory stimulation a delayed secondary polarization phase has frequently been seen, prolonging the inhibitory action. Repetitive inhibitory excitation may also be followed by a period of facilitation. Some examples of "direct" excitation by the depolarizing action of inhibitory impulses are described. 4. The interaction between antidromic and inhibitory impulses was studied. The results support previous conclusions (a) that during stretch the dendrites provide a persisting "drive" for the more central portions of the receptor cell, and (b) that antidromic all-or-none impulses do not penetrate into the distal portions of stretch-depolarized dendrites. The "after-potentials" of antidromic impulses are modified by inhibition. 5. Evidence is presented that inhibitory synaptic activity increases the conductance of the dendrites. This effect may occur in the absence of inhibitory potential changes.     

Expression and purification of a nanostructure-forming peptide

Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, P₁₁-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.     

Phosphoinositide breakdown in isolated rat parotid membranes. Stimulation by cholinergic and alpha-adrenergic agonists

Parotid gland membranes labelled with [3H]inositol were challenged with the cholinergic agonist, carbamylcholine, or with epinephrine in the presence of propranolol. Both agonists caused a significant breakdown of labelled phosphoinositides (17.5%) in membranes suspended in Krebs-Ringer bicarbonate buffer. This effect was abolished by the respective antagonists, atropine or phentolamine. The carbamylcholine-induced breakdown of labelled phosphoinositides did not require cytosol. The addition of cytosol alone, or the exposure of membranes to a medium of low ionic strength caused a significant breakdown of phosphoinositides (10-40%). No further breakdown due to the addition of carbamylcholine was observed under these conditions. It is suggested that neurotransmitter-induced breakdown of phosphoinositides is effected by membrane-associated enzyme(s) and can be observed only in a medium of high ionic strength.     

Immunoaffinity purification of SRT-tagged human creatine kinase by peptide elution

The mouse monoclonal antibody (Mab), SRT10, recognizes a linear epitope of 10 amino acids (ThrPheIleGlyAlaIleAlaThrAspThr). When these epitope-tagged fusion proteins are expressed in mammalian cells, the Mab can detect the tagged proteins by immunoblotting, immunocytochemistry and immunoprecipitation. Here, we describe an efficient method for the purification of SRT-tagged recombinant human creatine kinase (CK) transiently expressed in mammalian cells. This method utilizes the expression of the N-terminal- or C-terminal-tagged CK in transiently transfected HEK293 cells followed by binding to anti-SRT-agarose affinity resin and competitive elution with SRT peptide. Recombinant CK was purified near homogeneity as judged by SDS-PAGE.     

Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases

The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.     

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.     

Antihypertensive effect and purification of an ACE inhibitory peptide from sea cucumber gelatin hydrolysate

Gelatin from the sea cucumber (Acaudina molpadioidea) was hydrolyzed sequentially with bromelain and alcalase. The hydrolysate was fractionated into three ranges of molecular weight (GH-I, <10 kDa; GH-II, <5 kDa; GH-III, <1 kDa) using an ultrafiltration membrane bioreactor system. The GH-III brought about a high angiotensin-I-converting enzyme (ACE) inhibitory activity, with IC₅₀ value of 0.35 mg/ml. An ACE inhibitory peptide was isolated from the GH-III, using the chromatographic methods including ion-exchange chromatography, gel filtration and reversed phase high-performance liquid chromatography. The purified ACE inhibitory peptide with a molecular weight of 840 Da consisted of five main amino acids (Glu, Asp, Pro, Gly and Ala), and its IC₅₀ value was 0.0142 mg/ml. This sea cucumber gelatin hydrolysate (GH-III) was used as drinks administered to renal hypertensive rats (RHR) for 1 month. The systolic blood pressure and diastolic blood pressure of the RHR were significantly reduced, which indicates an antihypertensive effect by oral administration.     

Anticooperativity in a Glu-Lys-Glu salt bridge triplet in an isolated alpha-helical peptide

Salt bridges between oppositely charged side chains are well-known to stabilize protein structure, though their contributions vary considerably. Here we study Glu-Lys and Lys-Glu salt bridges, formed when the residues are spaced i, i + 4 surface of an isolated alpha-helix in aqueous solution. Both are stabilizing by -0.60 and -1.02 kcal/mol, respectively, when the interacting residues are fully charged. When the side chains are spaced i, i + 4, i + 8, forming a Glu-Lys-Glu triplet, the second salt bridge provides no additional stabilization to the helix. We attribute this to the inability of the central Lys to form two salt bridges simultaneously. Analysis of these salt bridges in protein structures shows that the Lys-Glu interaction is dominant, with the side chains of the Glu-Lys pair far apart.     

A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli

A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography. The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial activity assay.     

Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

BmK AngM1 is an analgesic peptide from the venom of Buthus martensii Karsch (BmK). The synthetic gene encoding BmK AngM1 was optimized on the basis of its cDNA sequence and the codon usage preference of Pichia pastoris. The codon-optimized gene was cloned into pPIC9K and then transformed into P. pastoris. SDS-PAGE and Western blot analysis showed that the recombinant BmK AngM1 (rBmK AngM1) was expressed by the addition of methanol to the medium, and its maximum production reached above 500 mg/l. The purified rBmK AngM1 could be obtained efficiently by Nickel affinity chromatography. Analgesic bioassay, by the mouse-twisting model, showed that rBmK AngM1 had evident analgesic effect with an ED₅₀ of 0.5 mg/kg.     

The breakdown of cell membranes by electrical and mechanical stress.

We attempted to determine whether mechanical tension and electrical stress couple to cause membrane breakdown in cells. Using cell-attached patches from HEK293 cells, we estimated the mechanically produced tension from the applied pressure and geometry of the patch. Voltage pulses of increasing amplitude were applied until we observed a sudden increase in conductance and capacitance. For pulses of 50 micros duration, breakdown required >0.5 V and was dependent on the tension. For pulses of 50-100 ms duration, breakdown required 0.2-0.4 V and was independent of tension. Apparently two physically different processes can lead to membrane breakdown. We could explain the response to the short, high-voltage pulses if breakdown occurred in the lipid bilayer. The critical electromechanical energy per unit area for breakdown by short pulses was approximately 4 dyne/cm, in agreement with earlier results on bilayers. Our data suggest that, at least in a patch, the bilayer may hold a significant fraction (approximately 40%) of the mean tension. To be compatible with the large, nonlytic area changes of patches, the bilayer appears to be pulled toward the pipette tip, perhaps by hydrophobic forces wetting membrane proteins bound to the glass. Although breakdown voltages for long pulses were in agreement with earlier work on algae, the mechanism(s) for this breakdown remain unclear.