108 results about "Periplasmic space" patented technology

Methods for the high yield production of antibody Fv-containing polypeptides, especially Fab′ and F(ab′)2 antibody fragments are provided. Expression of heavy and light chain Fv in a microbial secretory system is followed by recovery of Fv from the periplasm under conditions that maintain a cysteine residue as a free thiol. The free thiol is reacted with free thiol of an antibody fragment of the same or differing specificity, or with agents such as diagnostic labels or therapeutic moieties. The products offer advantages of homogeneity and purity not available through the use of known methods for preparing such derivatives.

A DNA encoding 20K hGH is connected directly to a gene encoding Escherichia coli OppA protein secretion signal, or a modified form thereof, and a DNA encoding signal peptidase 1 to construct a recombinant plasmid, E. coli is transformed by the plasmid and cells of the resulting E. coli transformant strain are cultured for secretory production of the 20K hGH in the E. coli periplasm. This method enables efficient secretory production of 20K hGH and easy isolation and purification of 20K hGH from the periplasm fraction because the level of impure proteins in the E. coli periplasm is low.

Compositions and methods for improving expression and / or secretion of protein or polypeptide of interest in a host cell are provided. Compositions comprising a coding sequence for a bacterial secretion signal peptide are provided. The coding sequences can be used in vector constructs or expression systems for transformation and expression of a protein or polypeptide of interest in a host cell. The compositions of the invention are useful for increasing accumulation of properly processed proteins in the periplasmic space of a host cell, or for increasing secretion of properly processed proteins from the host cell. In particular, isolated secretion signal peptide-encoding nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the nucleic acid molecules are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, and the nucleotide sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, as well as variants and fragments thereof.

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in the periplasm of gram negative bacteria and mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the outer surface-expression of ligand fusion proteins employed with phage display.

A bacterial high-expression system which is applicable for simultaneous screening of large numbers of recombinant clones from combinatorial antibody libraries is disclosed. The method pertains to screening of single chain antibodies from libraries expressed in the periplasm of E. coli by secretion. By this approach, approximately 104 clones can be screened in a single round. After screening, the clones, which express the recombinant antibodies to the desired antigen, can be directly used for production of large quantities of antibodies from microorganism culture. The system is especially attractive for fast screening of antibody libraries from a hybridoma source. A refolding method for the large-scale production of biologically active scFv-6 his proteins from bacterial inclusion bodies is also disclosed.

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating binding proteins capable of binding small molecules and peptides via “display-less” library screening. In the technique, libraries of candidate binding proteins, such as antibody sequences, are expressed in soluble form in the periplasmic space of gram negative bacteria, such as Escherichia coli, and are mixed with a labeled ligand. In clones expressing recombinant polypeptides with affinity for the ligand, the concentration of the labeled ligand bound to the binding protein is increased and allows the cells to be isolated from the rest of the library. Where fluorescent labeling of the target ligand is used, cells may be isolated by fluorescence activated cell sorting (FACS). The approach is more rapid than prior art methods and avoids problems associated with the surface-expression of ligand fusion proteins employed with phage display.

Genetically manipulated cells, lysates of such cells, systems, and methods of use thereof are provided, where one or more enzymes in a pathway of interest are genetically modified to incorporate a peptide sequence that provides for relocation of the protein, e.g., to the periplasm, so as to sequester the enzyme, and where the enzyme controls flux in the pathway of interest.

The present invention relates to the field of recombinant protein production in bacterial hosts. It further relates to expression of soluble, active recombinant protein by using secretion signals to direct the protein to the periplasmic space of a bacterial cell. In particular, the present invention relates to a production process for obtaining soluble hG-CSF protein from a bacterial host.