Eli Lilly and Company

Steven Kovacevic

James R Miller

Jeffrey T Fayerman

Paul Luther Skatrud

Thomas D Ingolia

Hansen M Hsiung

Mark A Richardson

Ramachandra N Rao

Loraine E Veal

Keith A Otto

John S Wood

Nancy E Beerman

The present invention provides novel fusion proteins comprising cyclins and CDKs. A preferred embodiment of the invention provides fusion proteins comprising human cyclin D1 and human CDK4. The fusion proteins of the invention optionally contain modifications, which facilitate their purification. Addition of histidine residues to selected constructs allows purification via immobilized metal affinity chromatography. Antigenic determinants allowing monoclonal antibody-based affinity chromatography purification are provided in selected embodiments of the invention. Protease cleavage sites are incorporated in selected constructs to allow cleavage of the regions incorporated in the cyclin-CDK fusion proteins for purification. Additional modifications which facilitate purification include strepavadin binding domains and antigenic determinants for antibody affinity chromatography.

Fusion proteins comprising cell cycle regulatory proteins

The present invention provides DNA compounds that encode deacetoxycephalosporin C synthetase (DAOCS) activity. The compounds can be used to construct recombinant DNA expression vectors for a wide variety of host cells, including E. coli, Penicillium, Streptomyces, Aspergillus, and Cephalosporium.

Recombinant DNA expression vectors and DNA compounds that encode

The present invention discloses a novel method for expressing and secreting a functional polypeptide in Bacillus including the vectors and transformants for the practice thereof. The vectors comprise the transcriptional and translational activity sequence, and optionally the signal peptide coding sequence, of the Staphylococcus aureus nuclease gene. The vectors and transformants are useful for the production and secretion of human hormones such as human proinsulin.

Method for expression and secretion in bacillus

A novel promoter, ribosome-binding site-encoding DNA, and a number of signal peptide-encoding DNA sequences are described for use in Bacillus and other host cells. The compounds are useful in constructing expression vectors for use in Bacillus and other host cells and drive expression and secretion of proteins such as nuclease and human growth hormone. In addition, a general method for obtaining cells with increased abilities to express and secrete heterologous gene products is described.

Vector comprising signal peptide-encoding DNA for use in Bacillus and

Recombinant DNA expression vectors for use in Bacillus and other host cells are disclosed. The vectors comprise the veg promoter sequence of Bacillus subtilis, a novel ribosome binding site-containing sequence and a sequence that codes for a functional polypeptide. The ribosome binding site-containing sequence is synthesized in accordance with conventional procedures while the veg promoter sequence can be obtained from E. coli K12 JA221/pMS480 (NRRL B-15258). Various sequences that codes for functional polypeptides and method for their expression in Bacillus are also disclosed.

Recombinant DNA expression vectors useful in bacillus and other host

A method for expressing a functional polypeptide in Streptomyces comprises transforming a Streptomyces host cell with a recombinant DNA expression vector and then culturing the transformed cell under conditions suitable for cell growth. The recombinant DNA expression vector comprises the veg or any other homologous Bacillus promoter, a naturally occurring or modified ribosome binding site-containing DNA sequence and a gene that codes for a functional polypeptide such as human pre-proinsulin. The method is specifically exemplified by use of expression plasmids pOW529, pOW539 and transformants, Streptomyces ambofaciens/pOW529 and Streptomyces ambofaciens/pOW539. The method is broadly applicable and is particularly useful in economically important Streptomyces taxa.

Method for using an homologous bacillus promoter and associated natural

The present invention discloses multifunctional recombinant DNA cloning vectors for use in Streptomyces, Bacillus, and E. coli. The invention further discloses transformants of the aforementioned vectors.

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Indianapolis, IN, United States of America

Steven Kovacevic