S100A4 interacts with p53 in the nucleus and promotes p53 degradation.

S100A4 is a small calcium-binding protein that is commonly overexpressed in a range of different tumor types, and it is widely accepted that S100A4 has an important role in the process of cancer metastasis. In vitro binding assays has shown that S100A4 interacts with the tumor suppressor protein p53, indicating that S100A4 may have additional roles in tumor development. In the present study, we show that endogenous S100A4 and p53 interact in complex samples, and that the interaction increases after inhibition of MDM2-dependent p53 degradation using Nutlin-3A. Further, using proximity ligation assay, we show that the interaction takes place in the cell nucleus. S100A4 knockdown experiments in two p53 wild-type cell lines, A549 and HeLa, resulted in stabilization of p53 protein, indicating that S100A4 is promoting p53 degradation. Finally, we demonstrate that S100A4 knockdown leads to p53-dependent cell cycle arrest and increased cisplatin-induced apoptosis. Thus, our data add a new layer to the oncogenic properties of S100A4 through its inhibition of p53-dependent processes.

Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

Negatively charged purification tags for selective anion-exchange recovery.

A novel strategy for the highly selective purification of recombinant fusion proteins using negatively charged protein domains, which were constructed by protein design, is described. A triple alpha-helical domain of 58 amino acids was used as scaffold. Far-ultraviolet circular dichroism measurements showed that the designed domains had very low alpha-helicity in a low-conductivity environment in contrast to the scaffold. The secondary structure could be induced by adding salt, giving a structure comparable to the parental molecule. Further studies showed that the new domains were able to bind to an anion exchanger even at pH values down to 5 and 6. Gene fusions between one of the designed domains and different target proteins, such as green fluorescent protein (GFP), maltose binding protein (MBP) and firefly luciferase, were also constructed. These gene products could be efficiently purified from whole cell lysates at pH 6 using anion-exchange chromatography.

In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor.

The 'detergent lipase' Lipolase, from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents. First it was demonstrated that wild-type Lipolase could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E. coli phage M13. A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed. Nine amino acids located in two regions close to the active site were targeted for randomization. Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase could be specifically enriched from a population of control phages. Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones. Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase-producing clone. In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase variants. Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant. The selected variants contained primarily basic amino acid residues within the other variegated region. Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.

Charge engineering of a protein domain to allow efficient ion-exchange recovery.

We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Zwt) was used as a scaffold when constructing two mutants, Zbasic1 and Zbasic2, with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Zwt. Although melting temperatures (Tm) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Zbasic1 and Zbasic2 showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Zbasic2 and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Zbasic2-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors.

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.

Production of a thermostable DNA polymerase by site-specific cleavage of a heat-eluted affinity fusion protein.

A novel strategy is described for bacterial expression and affinity purification of a recombinant truncated version of the heat-stable DNA polymerase I from Thermus aquaticus. The DNA polymerase (deltaTaq) was produced as a fusion to a serum albumin binding affinity handle (ABP) derived from streptococcal protein G. Based on the thermostability of the deltaTaq DNA polymerase, affinity-purified ABP-deltaTaq could be heat-eluted from HSA columns by incubation at 85 degrees C. To produce free deltaTaq DNA polymerase, efficient site-specific cleavage of the affinity tag was performed using a recombinant coxsackievirus 3C protease (3Cpro), also produced as an ABP affinity fusion. Thus, an integrated strategy could be devised where both the cleaved ABP affinity tag and the protease fusion could be recovered after site-specific cleavage using HSA-affinity chromatography. The flow-through fraction contained essentially pure deltaTaq DNA polymerase with full enzymatic activity.