Genentech, Inc.

John C Joly

Bradley Richard Snedecor

James Robert Swartz

Amy Shen

Benjamin Joseph Haley

Zhilan Hu

Dana Christian Andersen

The present invention provides compositions comprising a Fc-containing protein wherein substantially all the Fc domains have a C-terminal lysine. Further provided are host cell for producing said compositions, methods of making said host cells and compositions, and method of use thereof.

Expression of FC-containing proteins

Expression of Fc-containing proteins

A gram-negative bacterial cell is described that is deficient in a chromosomal gene present in a wild-type such cell which gene shares at least 80% sequence identity with the native sequence of the yfcK gene and encodes an aminopeptidase. Alternatively, a gram-negative bacterial cell is deficient in a chromosomal gene present in a wild-type such cell which gene encodes an aminopeptidase that shares at least 80% sequence identity with the native sequence of aminopeptidase b2324. Either of these types of cells, when comprising a nucleic acid encoding a heterologous polypeptide, produces an N-terminal unclipped polypeptide when it is cultured and the polypeptide recovered, with virtually no N-terminal clipped polypeptide produced as an impurity. Conversely, a method is provided for cleaving an N-terminal amino acid from a polypeptide comprising contacting the polypeptide with an aminopeptidase sharing at least 80% sequence identity with the native sequence of aminopeptidase b2324.

Method of using aminopeptidase to produce cleaved polypeptides

Method of N-terminal cleavage using an aminopeptidase

Protease-deficient cells

The invention provides a method for increasing product yield of a polypeptide of interest produced by recombinant host cells, where expression of the polypeptide by the recombinant host cells is regulated by an inducible system. More specifically, the method involves culturing the recombinant host cells under conditions of high metabolic and growth rate and then reducing the metabolic rate of the recombinant host cells at the time of induction of polypeptide expression. In particular, the invention provides a method of increasing product yield of an antibody, growth factor, or protease produced by a recombinant host cell regulated by an inducible system.

Metabolic rate shifts in fermentations expressing recombinant proteins

A process is provided for producing a heterologous polypeptide in bacteria. This process comprises, in a first step, culturing bacterial cells that lack their native pstS gene and comprise nucleic acid encoding a PstS variant having an amino acid variation within the phosphate-binding region of the corresponding native PstS, nucleic acid encoding a DsbA or DsbC protein, nucleic acid encoding the heterologous polypeptide, a signal sequence for secretion of both the DsbA or DsbC protein and the heterologous polypeptide, an inducible promoter for the nucleic acid encoding the DsbA or DsbC protein, and an alkaline phosphatase promoter for the nucleic acid encoding the heterologous polypeptide. The nucleic acid encoding a PstS variant is under the transcriptional control of the wild-type pstS gene promoter. The second step of the process involves recovering the heterologous polypeptide from the periplasm or the culture medium.

Process for bacterial production of polypeptides

A process is provided for producing a heterologous polypeptide in bacteria, which process comprises:

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San Mateo, CA, United States of America

John C Joly