Abstract: Microbial expression of an exogenous polypeptide may be inefficient if the messenger RNA has secondary structure (due to complementarity of spaced regions of the molecule) which impedes interaction with a ribosome. The occurrence of such structure can be predicted. The degeneracy of the genetic code gives freedom to alter the nucleic acid composition of the CDNA to eliminate harmful secondary structure of the MRNA.For example, bovine growth hormone (BGH) is poorly expressed by E. coli transformed with a recombinant plasmid containing the natural BGH gene. The corresponding MRNA has complementary regions at 46-51 and 73-78. Therefore a plasmid PBGH 33-4 was constructed containing a BGH gene whose upstream portion was synthesized (making use of plasmid PHGH 207-1 which contains an efficiently expressible gene for the closely related human growth hormone) so as not to cause the troublesome complementarity. E. coli transformed therewith produced BGH with-a yield of better than 10.sup.5 copies per cell.

Abstract: Novel single-chain protease resistant urokinase derivatives are provided. In particular, derivatives are provided wherein the Lys.sub.135 Lys.sub.136 and Arg.sub.156 to Lys.sub.158 sites are rendered less susceptible to proteolytic cleavage are provided by occluding the sites or by covalently modifying them. Preferred covalent modifications are amino acid sequence variants at the sites where proteolysis of urokinase occurs. These are optimally produced by synthesis of single-chain urokinase mutants in recombinant cell culture. The novel urokinase derivatives herein offer the advantage of avoiding the generation of substantial two-chain urokinase, either in vivo or during recombinant cell culture. However, the derivatives continue function to activate plasminogen in initiating blood clot lysis.

Abstract: Biologically active mutant tissue plasminogen activators are disclosed wherein site directed mutagenesis, for example, of a two-chain activation site renders the mutants resistant to conversion to the two-chain form.

Abstract: Human urokinase is produced using recombinant DNA techniques. The invention disclosed thus enables the production of urokinase free of contaminants with which it is ordinarily associated in its native cellular environment. Methods, expression vehicles and various host cells useful in its production are also disclosed.

Abstract: Biologically active mutant tissue plasminogen activators are disclosed wherein site directed mutagenesis, for example, of a two-chain activation site renders said mutants resistant to conversion to the two-chain form.

Abstract: Direct linkage of DNA encoding prokaryotic signals such as E. coli enterotoxin with DNA encoding mature eukaryotic proteins followed by transformation and culture of bacterial hosts characterized by a periplasmic space, yields substantial amounts of periplasmic mature protein.

Abstract: Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.

Abstract: The production of mature hGH in E coli and Pseudomonas strains transformed by a plasmid which encodes pre hGH (comprising the signal polypeptide and the hormone itself) is described. These prokaryotes process the pre-hGH to cleave the signal sequence and, thereby, produce mature hGH.

Abstract: Altered and native immunoglobulins, including constant-variable region chimeras, are prepared in recombinant cell culture. The immunoglobulins contain variable regions which are immunologically capable of binding predetermined antigens. Methods are provided for refolding directly expressed immunoglobulins into immunologically active form.

Abstract: The production of mature hGH In E. coli and Pseudomonas strains transformed by a plasmid which encodes pre hGH (comprising the signal polypeptide and the hormone itself) is described. These prokaryotes process the pre-hGH to cleave the signal sequence and, thereby, produce mature hGH.

Abstract: A method of altering double stranded DNA comprising converting double stranded DNA to single stranded DNA in a region surrounding a selected cleavage site, hybridizing a complimentary primer to the single stranded region formed in the previous step, and restoring enzymatically the second strand which had been eliminated.

Abstract: Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.

Abstract: Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.

Abstract: Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.

Abstract: Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.

Abstract: Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.