Ribonuclease-charged vector for facile direct cloning with positive selection

Plasmid vectors for positive selection of cloned inserts in Escherichia coli were devised, based on an expression plasmid (pMT416) for the bacterial ribonuclease barnase. In addition to the barnase gene under control of a synthetic tac promoter, these plasmids carry the gene for the barnase inhibitor, barstar, the constitutive expression of which protects the bacterium from the detrimental effects of moderate barnase production. Full expression of the barnase gene overcomes protection by barstar and becomes lethal. Having a unique SmaI/XmaI site in the barnase structural gene, pMT416 itself can be used as a selective vector: uncut or religated pMT416 will preclude growth while plasmids with inserts in the barnase gene will allow the cells to survive. The entire pUC polylinker was inserted into the barnase gene in place of the Val-36 codon. This insert of nineteen largely hydrophilic amino acids does not prevent the lethal effect of full expression of the gene. The resulting plasmid, pMT440, is a generally useful selective cloning vector representing the “kill-the-rest” approach. Received: 25 April 1998 / Accepted: 26 May 1998     

核糖核酸酶barnase基因的克隆策略研究

比较了目前报道的两种克隆barnase基因的方法——barnase基因的单独克隆和barnase抑制剂基因保护下的barnase基因克隆。分别利用4种质粒进行barnase基因的单独克隆时,得到的阳性克隆数量少。对其中15个阳性克隆进行了测序分析,结果有5个克降出现碱基缺失,1个克隆出现碱基增加,其余9个克隆则出现氨基酸突变,均未得到氨基酸序列完正确的克隆。进一步分析表明这些突变的位点大多直接与保持barnase蛋白的活性位点相关。而在克隆barnase基因之前,预先将barnase基因连同其自身启动了加载于待克隆载体上,随后再进行barnase基因克隆时则容易得到与报道的barnase基因序列完全一致的阳性克隆。分析认为由于barnase基因单独存在时有一定数世的蛋白表达,宿主菌大肠杆菌无法忍受而通过某种方式造成其barnase发生相应突变而无法实现barnase基因的单独克隆。因此有必要利用barnase基因的保护来进行barnase基因相应的克降和遗传操作。     

A novel strategy for regulated expression of a cytotoxic gene.

The tetracycline (Tet) transactivator system is a powerful promoter system to control gene expression. However, expression of a cytotoxic gene in this system has been limited due to the lethal effect caused by low levels of basal expression of the toxic gene. In this report, we describe a novel strategy to express a toxic gene using the Tet system. The barstar gene is placed downstream of a minimal promoter and the barnase gene downstream of the tetracycline responsive element minimal promoter. When barnase is expressed at a basal level, its toxicity in human cell culture is offset by the similar basal level expression of barstar. However, when the barnase expression is induced with the transactivator protein, its overproduction leads to cell death. Therefore, this strategy allows cytotoxicity to be effectively regulated by tetracycline.     

A novel cell ablation strategy blocks tobacco anther dehiscence.

We utilized a new cell ablation strategy to ablate specific anther cell types involved in the dehiscence process. The tobacco TA56 gene promoter is active within the circular cell cluster, stomium, and connective regions of the anther at different developmental stages. We introduced a cytotoxic TA56/barnase gene into tobacco plants together with three different anticytotoxic barstar genes. The anticytotoxic barstar genes were used to protect subsets of anther cell types from the cytotoxic effects of the TA56/barnase gene. The chimeric barstar genes were fused with (1) the tobacco TP12 gene promoter that is active at high levels in most anther cell types; (2) the soybean lectin gene promoter that is active earlier in the connective, and at lower levels in the circular cell cluster and stomium, than is the TA56 promoter; and (3) the tobacco TA20 gene promoter that is active at high levels in most anther cell types but has a different developmental profile than does the TP12 promoter. Normal anther development and dehiscence occurred in plants containing the TA56/barnase and TP12/barstar genes, indicating that barstar protects diverse anther cell types from the cytotoxic effects of barnase. Anthers containing the TA56/barnase and lectin/barstar genes also developed normally but failed to dehisce because of extensive ablation of the circular cell cluster, stomium, and contiguous connective regions. Anthers containing the TA56/barnase and TA20/barstar genes failed to dehisce as well. However, only the stomium region was ablated in these anthers. The connective, circular cell cluster, and adjacent wall regions were protected from ablation by the formation of barnase/barstar complexes. We conclude that anther dehiscence at flower opening depends on the presence of a functional stomium region and that chimeric barnase and barstar genes containing promoters that are active in several overlapping cell types can be used for targeted cell ablation experiments.     

The cloning and expression of the RNAse structural gene of Bacillus intermedius]

The structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity. Using biochemical immunochemical analysis the expression of binase gene in E. coli cells has been confirmed. The recombinant clone E. coli which contains both plasmids simultaneously--carrying gene for barster and for benase has been produced. The given vector is suggested to be used for cloning of inhibitor gene to obtain a viable producer of alkaline intracellular ribonuclease.     

Barnase and barstar: two small proteins to fold and fit together

Barnase and barstar are the extracellular ribonuclease and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Both are small single-chain proteins and thus are suitable for application to the study of how a protein's sequence directs its fold. Barnase has neither disulfide bonds nor non-peptide components and unfolds reversibly in what closely approximates a two-state reaction. The genes for both these proteins have been cloned in E. coli. Expression of barstar is necessary to counter the lethal effect of expressed active barnase. Site-directed mutagenesis is being used to answer specific and general questions relating to protein folding and protein-protein interaction.     

禾谷类作物雄性不育及育性恢复表达载体的构建

通过ＰＣＲ反应扩增出了玉米花药特异启动子ＣＡ５５,将其分别与Ｂａｒｎａｓｅ和Ｂａｒｓｔａｒ基因融合,构建成了植物雄性不育基因ＣＡ５５ＢＮＮＯＳ和其育性恢复基因ＣＡ５５ＢＳＮＯＳ,再将它们分别插入到ｐＣＡＭＢＩＡ３３００中,获得了应用于禾谷类作物的基因工程雄性不育及育性恢复的表达载体。     

Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex.     

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His(5)) with an NH(2)-terminal leader peptide. The 4D5 scFv-barnase-His(5) gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His(5) fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His(5) on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.     

Design of multivalent complexes using the barnase*barstar module

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185(HER2-ECD) 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.     

Downhill binding energy surface of the barnase–barstar complex

We have employed biased molecular dynamics simulations in explicit solvent to characterize the one‐dimensional potential of mean force for the dissociation process of the barnase–barstar protein–protein complex. Unbinding of barstar from wild‐type barnase was compared with dissociation from four charge‐deletion mutants of barnase. Interestingly, we find in all cases that unbinding of barnase and barstar is an uphill process on a smooth, tilted energy landscape. The total free energy difference between the dissociated and bound state was similar for wild‐type barnase–barstar and for the R87A mutant of barnase. The values for the three other mutant barnase mutants K27A, R59A, and R83Q were only about half as much. Besides, we have analyzed the conformational dynamics of important residues at the barnase–barstar interface. In the bound state, their conformational fluctuations are reduced relatively to the free state because of the formation of intermolecular contacts. Interestingly, we find that some residues also show decreased mobility at intermediate stages of the unbinding process suggesting that these residues may be involved in the first contacts being formed on binding. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 977–985, 2010.     

X-ray structural analysis of compensating mutations at the barnase-barstar interface.

The crystal structure of the barstar mutants (Y29P) and (Y29D, Y30W) as well as that of the complexes of barstar(Y29P) with wild-type barnase and barnase(H102K) have been determined. These barstar mutants compensate for the dramatic loss of barnase-barstar interaction energy caused by a single mutation of the barnase active site His-102 to a lysine. The latter introduces an uncompensated charge in the pocket at the surface of barstar where Lys-102 is located. The analysis of the structures suggests a mechanism for this compensation based on the solvation of the charge of Lys-102. Additional compensation occurs through the formation of a hydrogen bond.     

Barstar is electrostatically optimized for tight binding to barnase

We used a novel charge optimization technique to study the small ribonuclease barnase and to analyze its interaction with a natural tight binding inhibitor, the protein barstar. The approach uses a continuum model to explicitly determine the charge distributions that lead to the most favorable electrostatic contribution to binding when competing desolvation and interaction effects are included. Given its backbone fold, barstar is electrostatically optimized for tight binding to barnase when compared with mutants where residues have been substituted with one of the 20 common amino acids. Natural proteins thus appear to use optimization of electrostatic interactions as one strategy for achieving tight binding.     

Bisexual sterility conferred by the differential expression of Barnase and Barstar: a simple and efficient method of transgene containment

To establish a simple and an efficient system to minimize the environmental risk of genetically modified plants, we tested the applicability of the barnase/barstar system in conferring bisexual sterility; that is, in preventing plants setting seeds by self-fertilization and out-crossing. Transgenic tobacco plants were generated to express barnase, a cell death inducing ribonuclease, under the control of the gamete-specific AtDMC1 promoter, and barstar, a specific inhibitor of barnase, under the control of the ACT2 promoter, which is constitutively active in almost all tissues except gametes. In contrast to control plants harboring the barstar expression unit only, which set seeds normally with self-pollination, all transformants harboring both barnase and barstar were bisexually sterile. They produced aberrant anthers containing no detectable pollen and failed to set seeds even after pollination with wild-type tobacco pollen.An erratum to this article can be found at     

Use of plasmid pTV1 in transposon mutagenesis and gene cloning in Bacillus amyloliquefaciens

The plasmid pTV1, constructed in Bacillus subtilis as a tool for insertional mutagenesis by the transposon Tn917, has been transferred to Bacillus amyloliquefaciens by transduction with the phage PBS1. Insertional mutants containing Tn917 were observed in the new host. Southern blot analysis of such mutants indicated no preference for insertion sites. The copy numbers of pTV1 in B. amyloliquefaciens and B. subtilis were found to be 1.4 and 14, respectively; the plasmid is less stable against loss in B. amyloliquefaciens. The overall transposition rate in B. amyloliquefaciens is nevertheless comparable to that in B. subtilis and large numbers of mutants are readily obtained. The yield of auxotrophs was about 0.7% of all mutants, but the preponderance of glutamate auxotrophs seen in B. subtilis was not observed. A number of auxotrophs were identified as to nutritional requirements and those tested were found to be stable. Mutants deficient in extracellular proteases, amylase, and ribonuclease (barnase) were also found and the inactivated barnase gene has been cloned in Escherichia coli. It seems likely, therefore, that any B. amyloliquefaciens gene for which there is a functional test could be cloned by this technique.     

Diffusional encounter of barnase and barstar

We present an analysis of trajectories from Brownian dynamics simulations of diffusional protein-protein encounter for the well-studied system of barnase and barstar. This analysis reveals details about the optimal association pathways, the regions of the encounter complex, possible differences of the pathways for dissociation and association, the coupling of translational and rotation motion, and the effect of mutations on the trajectories. We found that a small free-energy barrier divides the energetically most favorable region into a region of the encounter complex above the barnase binding interface and a region around a second energy minimum near the RNA binding loop. When entering the region of the encounter complex from the region near the RNA binding loop, barstar has to change its orientation to increase the electrostatic attraction between the proteins. By concentrating the analysis on the successful binding trajectories, we found that the region of the second minimum is not essential for the binding of barstar to barnase. Nevertheless, this region may be helpful to steer barstar into the region of the encounter complex. When applying the same analysis to several barnase mutants, we found that single mutations may drastically change the free-energy landscape and may significantly alter the population of the two minima. Therefore, certain protein-protein pairs may require careful adaptation of the positions of encounter and transition states when interpreting mutation effects on kinetic rates of association and/or dissociation.     

Interaction energy decomposition in protein-protein association: a quantum mechanical study of barnase-barstar complex

Protein-protein interactions are very important in the function of a cell. Computational studies of these interactions have been of interest, but often they have utilized classical modelling techniques. In recent years, quantum mechanical (QM) treatment of entire proteins has emerged as a powerful approach to study biomolecular systems. Herein, we apply a semi-empirical divide and conquer (DC) methodology coupled with a dielectric continuum model for the solvent, to explore the contribution of electrostatics, polarization and charge transfer to the interaction energy between barnase and barstar in their complex form. Molecular dynamic (MD) simulation was performed to account for the dynamic behavior of the complex. The results show that electrostatics, charge transfer and polarization favor the formation of the complex. Our study shows that electrostatics dominates the interaction between barnase and barstar ( approximately 73%), while charge transfer and polarization are approximately 21% and approximately 6%, respectively. Close inspection of the polarization and charge-transfer effects on the charge distribution of the complex reveals the existence of two, well localized, regions in barstar. The first region includes the residues between P27 and Y47 and the second region is between N65 and D83. Since no such regions could be detected in barnase clearly suggests that barstar is well optimized for efficiently binding barnase. Furthermore, using our interaction energy decomposition scheme, we were able to identify all residues that have been experimentally determined to be important for the complex formation and to suggest other residues never have been investigated. This suggests that our approach will be useful as an aid in further understanding protein-protein contacts for the ultimate goal to produce successful inhibitors for protein complexes.     

Propagation of dynamic changes in barnase upon binding of barstar: an NMR and computational study

NMR spectroscopy and computer simulations were used to examine changes in chemical shifts and in dynamics of the ribonuclease barnase that result upon binding to its natural inhibitor barstar. Although the spatial structures of free and bound barnase are very similar, binding results in changes of the dynamics of both fast side-chains, as revealed by (2)H relaxation measurements, and NMR chemical shifts in an extended beta-sheet that is located far from the binding interface. Both side-chain dynamics and chemical shifts are sensitive to variations in the ensemble populations of the inter-converting molecular states, which can escape direct structural observation. Molecular dynamics simulations of free barnase and barnase in complex with barstar, as well as a normal mode analysis of barnase using a Gaussian network model, reveal relatively rigid domains that are separated by the extended beta-sheet mentioned above. The observed changes in NMR parameters upon ligation can thus be rationalized in terms of changes in inter-domain dynamics and in populations of exchanging states, without measurable structural changes. This provides an alternative model for the propagation of a molecular response to ligand binding across a protein that is based exclusively on changes in dynamics.     

Characterization of in vitro oxidized barstar

The polypeptide inhibitor of the ribonuclease barnase, barstar, has two cysteine residues in positions 40 and 82. These have been proposed to form a disulfide bridge leading to an increase in stability without changing the inhibitory activity of the protein. Barstar and a mutant (E80A) were oxidized in vitro and the biochemical and physico-chemical properties of the oxidized monomers were analysed. The oxidized proteins show no inhibition of barnase using a plate assay and are significantly destabilized. CD spectra indicate a loss of secondary structure. The amino acid substitution E80 → A stabilizes the oxidized barstar to about the same extent as it does the reduced protein, indicating, however, that the helical region which it is in is intact.