ABSTRACT
Bacillus subtilis spores are considered to be efficient and useful vehicles for the surface display and delivery of heterologous proteins. In this study, we prepared recombinant spores with the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein displayed on their surface in fusion with the CotZ or CotY spore coat proteins as a possible tool for the development of an oral vaccine against the SARS-CoV-2 virus. The RBD was attached to the N-terminus or C-terminus of the coat proteins. We also directly adsorbed non-recombinantly produced RBD to the spore surface. SDS-PAGE, western blot and fluorescence microscopy were used to analyze RBD surface expression on purified spores. Results obtained from both display systems, recombinant and non-recombinant, demonstrated that RBD was present on the spore surfaces.
ABSTRACT
RNase Sa3 produced by Streptomyces aureofaciens strain CCM 3239 belongs to the T1 family of microbial ribonucleases. It is closely related both to RNase Sa, studied in detail earlier, and to RNase Sa2 produced by the same microorganism. The most important property of RNase Sa3 is the relatively high cytotoxic activity, which was not observed for RNase Sa and Sa2. Recombinant RNase Sa3 was overexpressed in Escherichia coli and purified to high homogeneity. The hanging-drop vapour-diffusion method was used for crystallization. The two crystal forms are trigonal P3(1)21 and tetragonal P4(1)2(1)2, with unit-cell parameters a = b = 64.7, c = 69.6 A, gamma = 120 degrees and a = b = 34.0, c = 147.2 A, respectively. They diffract to 2.0 and to 1.7 A resolution, respectively, using synchrotron radiation. The asymmetric units of crystal forms I and II contain one molecule of the enzyme, which corresponds to V(M) = 3.8 A(3) Da(-1) with a solvent content of 68% and V(M) = 1.9 A(3) Da(-1) with a solvent content of 37%, respectively.
Subject(s)
Isoenzymes/chemistry , Ribonucleases/chemistry , Crystallization , Crystallography, X-Ray , Isoenzymes/isolation & purification , Protein Conformation , Ribonucleases/isolation & purification , Streptomyces aureofaciens/enzymologySubject(s)
Enzyme Inhibitors/chemistry , Isoenzymes/antagonists & inhibitors , Ribonucleases/antagonists & inhibitors , Streptomyces aureofaciens/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Biophysical Phenomena , Biophysics , Enzyme Inhibitors/isolation & purification , Isoenzymes/chemistry , Macromolecular Substances , Ribonucleases/chemistryABSTRACT
Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces aureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, Tm, at pH 7 of 48.4 degrees C (Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The Tm values are increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The Tm values are decreased by 20.0 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two other members of the microbial ribonuclease family, RNase T1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2), 5.6 (Sa3), 6.1 (Sa), 6.6 (T1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these proteins.
Subject(s)
Isoenzymes/chemistry , Protein Denaturation/drug effects , Protein Folding , Ribonucleases/chemistry , Streptomyces aureofaciens/chemistry , Amino Acid Sequence , Disulfides/chemistry , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino Acid , Sodium Chloride/pharmacology , Temperature , Thermodynamics , Urea/pharmacologyABSTRACT
Two new RNase inhibitors, SaI14 (Mr, approximately 14,000) and SaI20 (Mr, approximately 20,000), were isolated and purified from a Streptomyces aureofaciens strain. The gene sai14, coding for SaI14 protein, was cloned and expressed in Escherichia coli. The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor from Bacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.
