J.M. Mancheño

Overproduction and purification of biologically active native fungal α-sarcin in Escherichia coli

An efficient system was developed to produce, in Escherichia coli, large amounts of native alpha-sarcin (alpha Sar), a cytotoxin from the mold Aspergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 micrograms/ml of original culture. The constructed expression vector (pINPG alpha S) is based on the synthesis of a fusion protein between alpha Sar and a modified version of the OmpA signal peptide. This peptide seems to favour the postranslational processing of the fusion protein. The purified recombinant alpha-sarcin (re-alpha Sar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, chromatographic behaviour, immunoreactivity and spectroscopic features. Indeed, the recombinant protein recovered is completely functional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal alpha Sar.

MEMBRANE INTERACTION OF A BETA -STRUCTURE-FORMING SYNTHETIC PEPTIDE COMPRISING THE 116-139TH SEQUENCE REGION OF THE CYTOTOXIC PROTEIN ALPHA -SARCIN

alpha-Sarcin is a cytotoxic protein that strongly interacts with acid phospholipid vesicles. This interaction exhibits a hydrophobic component although alpha-sarcin is a highly polar protein. A peptide comprising the amino acid sequence corresponding to the 116-139th segment of the alpha-sarcin cytotoxin has been synthesized by a standard fluoren-9-yl-methoxycarbonyl-based solid phase method. Its primary structure is: (116)-NPGPARVIYTYPNKVFCGIIAHTK-(139). Two beta-strands have been predicted in this region of alpha-sarcin, where the less polar stretches of the protein are found. The synthetic peptide interacts with negatively charged large unilamellar vesicles of either natural or synthetic phospholipids. An apparent fragmentation of the vesicles is produced by the peptide based on electron microscopy studies. The peptide promotes leakage of the intravesicular aqueous contents and lipid mixing of bilayers. The packing of the phospholipid molecules is greatly perturbed by the peptide, as deduced from the drastic changes induced by the peptide in cooperative properties associated with the phase transition of the bilayers. At saturating peptide/phospholipid ratios, the phase transition of dimyristoylphosphatidylglycerol vesicles is abolished. All of these effects are saturated at about 0.3 peptide/lipid molar ratio. The peptide adopts a mostly random structure in aqueous solution. A conformation composed of a high proportion of antiparallel beta-sheet is induced as a consequence of the interaction with the phospholipid vesicles in opposition to trifluoroethanol that promotes alpha-helical peptide structures, as deduced from circular dichroism measurements. The obtained results are discussed in terms of the potential involvement of the region comprising residues 116-139 of alpha-sarcin in the hydrophobic interactions of this cytotoxic protein with membranes.

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles. The dilution of the fluorescence probes was faster when these were incorporated into the bilayers made of synthetic phospholipids than were present in those made of natural phospholipids. The bilayer destabilization produced by the protein in the vesices composed of the dimyristoyl-phospholipids should be transmitted to the more stable ones made of natural phospholipids. The obtained results are interpreted in terms of lipid mixing occurring within vesicle aggregates larger than dimer.

Spectroscopic characterization of the alkylated α-sarcin cytotoxin: analysis of the structural requirements for the protein-lipid bilayer hydrophobic interaction

alpha-Sarcin is a ribosome-inactivating protein that translocates across lipid bilayers, these two abilities explaining its cytotoxic character. This protein is composed of a single polypeptide chain with two disulfide bridges. Reduction and carboxyamidomethylation of alpha-sarcin results in protein unfolding, based on the results of the spectroscopic characterization of the chemically modified protein. The absorption and fluorescence emission bands of the tryptophan residues of the modified protein appear blue- and red-shifted, respectively. Far-UV circular dichroism analysis reveals the presence of residual secondary structure (beta-strands and turns) in the alkylated protein. This retains its ability to interact with lipid bilayers. It promotes vesicle aggregation, lipid-mixing between bilayers and leakage of the intravesicular aqueous contents. The modified protein tends to abolish the phase transition of acid phospholipids as detected by differential scanning calorimetry and depolarization measurements of fluorescence-labelled vesicles. The protein gain access to vesicle-entrapped trypsin. The fluorescence emission of the tryptophan residues is blue-shifted upon interaction of the protein with the bilayers, and anthracene incorporated into the hydrophobic core of the membranes quenches the tryptophan fluorescence emission of the protein. The secondary structure of the alkylated protein interacting with lipid vesicles has been studied by infrared spectroscopy. An increase in the alpha-helix and turn contents and a concomitant decrease in the beta-structure content are observed upon interaction with the bilayers. The results obtained are discussed in terms of the structural requirements for the interaction of alpha-sarcin with lipid membranes.

Escherichia coli JA221 can suppress the UAG stop signal

The Escherichia coli strain JA221 can suppress the UAG stop codon, although the existence of an amber suppressor tRNA has not previously been described for this strain. When using a plasmid to express α‐sarcin, which has TAG as its stop signal, two proteins were obtained: a smaller protein corresponding in size to that of the expected protein, and a larger protein, which could be accounted for by the presence of a second stop codon (TGA) 18 base pairs downstream of the original. This feature of strain JA221 must therefore be considered when using this strain as a host for the production of recombinant proteins.