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

Several plasmids which are useful for genetically transforming plant cells. A first plasmid, such as pMON120, contains a T-DNA border, one or more marker genes, a unique cleavage site, and a region of Ti plasmid homology. A gene which is expressed in plant cells may be inserted into this plasmid to obtain a derivative plasmid, such as pMON128 which expresses neomycin phosphotransferase in plant cells. The derivative plasmid is inserted into a suitable microorganism, such as A. tumefaciens which contains a Ti plasmid. The inserted plasmids recombine with the Ti plasmids to form co-integrate plasmids. Only a single crossover event is required to create the desired co-integrate plasmid. A. tumefaciens cells with co-integrate plasmids are selected and co-cultured with plant cells. The co-integrate Ti plasmids enter the plant cells and insert a segment of T-DNA which does not contain tumorigenic genes into the plant genome. The transformed plant cell(s) may be regenerated into a morphologically normal plant which will pass the inserted gene(s) to its descendants.

Plasmids for transforming plant cells

A method for identifying a microorganism having a reduced adaptation to a particular environment comprising the steps of: (1) providing a plurality of microorganisms each of which is independently mutated by the insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence so that each mutant contains a different marker sequence, or clones of the said microorganism; (2) providing individually a stored sample of each mutant produced by step (1) and providing individually stored nucleic acid comprising the unique marker sequence from each individual mutant; (3) introducing a plurality of mutants produced by step (1) into the said particular environment and allowing those microorganisms which are able to do so to grow in the said environment; (4) retrieving microorganisms from the said environment or a selected part thereof and isolating the nucleic acid from the retrieved microorganisms; (5) comparing any marker sequences in the nucleic acid isolated in step (4) to the unique marker sequence of each individual mutant stored as in step (2); and (6) selecting an individual mutant which does not contain any of the marker sequences as isolated in step (4).

Identification of genes

Disclosed are methods of producing eukaryotic disulfide bond-containing polypeptides in bacterial hosts, and compositions resulting therefrom. Co-expression of a eukaryotic foldase and a disulfide bond-containing polypeptide in a bacterial host cell is demonstrated. In particular embodiments, the methods have been used to produce mammalian pancreatic trypsin inhibitor and tissue plasminogen activator (tPA) in soluble, biologically-active forms, which are isolatable from the bacterial periplasm. Also disclosed are expression systems, recombinant vectors, and transformed host cells.

Methods for producing soluble, biologically-active disulfide-bond containing eukaryotic proteins in bacterial cells

Disclosed are methods and compositions for producing heterologous disulfide bond containing polypeptides in bacterial cells. In preferred embodiments, the methods involve co-expression of a prokaryotic disulfide isomerase, such as DsbC or DsbG and a gene encoding a recombinant eukaryotic polypeptide. Exemplary polypeptides disclosed include tissue plasminogen activator.

Methods for producing heterologous disulfide bond-containing polypeptides in bacterial cells

Microorganism having a gene derived from a higher organism is produced by isolating cells from a higher organism containing messenger RNA, extracting the messenger RNA, synthesizing a double stranded cDNA using the messenger RNA as a template, inserting the cDNA into a plasmid and transforming a microorganism with the resultant recombinant plasmid.

Method for producing recombinant bacterial plasmids containing the coding sequences of higher organisms

The disclosure sets forth a process for isolating a specific nucleotide sequence containing genetic information, synthesis of DNA having the specific nucleotide sequence and transfer of the DNA to a host microorganism The process involves isolation of a selected cell population, extraction of intact mRNA from the cells, purification of intact messenger RNA from the extract and subjection of the purified extract to the action of reverse transcriptase in the presence of the four deoxynucleoside triphosphates needed to synthesize a complementary (cDNA) strand. The ribonucleotide sequence is selectively removed and the remaining deoxynucleotide sequence is incubated with reverse transcriptase or DNA polymerase in the presence of the four deoxynucleoside triphosphates to form a duplex cDNA structure having its comple menary strands joined together at one end by a single stranded loop. This product then is treated with single strand specific nuclease to cleave the single stranded loop, the resulting double stranded cDNA is extended in length by the addition at both ends of a specific DNA containing a restriction enzyme recognition site sequence, and the extended cDNA is treated with a restriction endonuclease to produce self-complementary single stranded ends at the five-prime termini of each strand in the duplex. A plasmid DNA having a recognition site for the same endonuclease is treated with the enzyme to cleave the polynucleotide strand, the 5' terminal phosphate groups from the resulting self-complementary single strand nucleotide sequences at the five-prime termini are removed and the prepared cDNA and plasmid DNA are incubated in the presence of DNA ligase, so that a viable closed ring of plasmid DNA can only occur if a segment of cDNA is included the cDNA-containing plasmid then being introduced to an appropriate host cell. Pure bacterial strains containing the recombinant plasmid are grown up and isolated. By this procedure, large amounts of recombinant plasmid DNA may be prepared and the specific cDNA sequence reisolated therefrom by endonucleolytic cleavage.

Recombinant bacterial plasmids containing the coding sequences of insulin genes

The structure and expression of the insulin gene

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Method for producing recombinant bacterial plasmids containing the coding sequences of higher organisms

Recombinant bacterial plasmids containing the coding sequences of insulin genes

The structure and expression of the insulin gene