Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli.

The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disorders or conditions using albumin fusion proteins of the invention.

Albumin Fusion Proteins

Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.

/pdf/a-novel-secretory-pathway-for-interleukin-1-beta-a-protein-gblt158qcv.pdf

A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence.

Transglutaminases: protein cross-linking enzymes in tissues and body fluids.

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of β1 and β3 subfamilies, but not with β2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

https://rupress.org/jcb/article-pdf/148/4/825/1290576/9909135.pdf

Tissue Transglutaminase Is an Integrin-Binding Adhesion Coreceptor for Fibronectin

Complete cDNA sequence encoding human β-galactoside α-2, 6-sialyltransferase

A cDNA library prepared from human placenta has been screened for sequences coding for factor XIIIa, the enzymatically active subunit of the factor XIII complex that stabilizes blood clots through crosslinking of fibrin molecules. Two oligonucleotides, based on the amino acid sequences of tryptic peptides of factor XIIIa, were used as hybridization probes. Of 0.36 X 10(6) independent recombinants, 1 clone was identified that hybridized to both probes. The insert of 1704 base pairs coded for the amino-terminal 541 amino acid residues of the mature factor XIIIa molecule. Blot-hybridization analysis using this cDNA as a probe showed that the factor XIIIa mRNA from placenta has a size of approximately 4000 bases. The insert was used to rescreen cDNA libraries and to identify further factor XIIIa-specific sequences. The total length of the isolated factor XIIIa cDNA is 3905 bases, and it codes for a protein of 732 amino acids. In spite of the presence of factor XIII in blood plasma, we could not identify a leader sequence typical for secreted proteins.

Characterization of cDNA coding for human factor XIIIa

A mutant of factor VIII : C in which the amino acids Ser to Arg are absent from the new molecule consequently retains of the 4 thrombin cleavage sites which have hitherto been regarded as essential only the cleavage site at Arg . The cleavage site at Arg is absent, and the amino acids adjacent to the cleavage sites at Arg and Arg are present, and linkage thereof produces a new cleavage site. The protein has a procoagulant activity and biological half-life very similar to that of the authentic protein and, moreover, is more rapidly activated by thrombin.

Factor viii:c-like molecule with a coagulant activity

The recent cloning and sequence analysis of human factor VIII:C (antihaemophilic factor) revealed a domain structure for the protein which can be presented as A1-A2-B-A3-C1-C2. In this report we describe the construction of two altered factor VIII:C cDNAs coding for molecules in which a part (amino acids 816 to 1598) or all of the B domain (amino acids 741 to 1689) was removed. In the latter mutant a new thrombin cleavage site has been created, which does not exist in wild-type factor VIII:C. The mutated cDNAs were cloned into eucaryotic expression vectors based on regulatory sequences of the virus SV40 and transfected into two different mammalian cell lines. Both truncated recombinant factor VIII:C molecules were secreted into the culture medium, showed full biological activity and could be activated by thrombin.

Synthesis of biologically active deletion mutants of human factor VIII:C.

The cDNA which encodes the factor XIIIa was isolated with the aid of a cDNA library of human placenta and probes which had been constructed following the amino acid sequence of factor XIIIa peptide fragments. Not only does this cDNA allow the factor XIIIa to be obtained by genetic engineering in high purity but it also allows the preparation of diagnostics which permit the analysis of genetically determined factor XIIIa defects. Moreover, antibodies which are suitable for diagnostics or antibody columns can be prepared on the basis of the amino acid sequence.

Gerd Dr. Zettlmeissl

Papers

Complete cDNA sequence encoding human β-galactoside α-2, 6-sialyltransferase

Characterization of cDNA coding for human factor XIIIa

Factor viii:c-like molecule with a coagulant activity

Synthesis of biologically active deletion mutants of human factor VIII:C.

Production of factor XIIIa by gene technology