Novel signal sequences to improve protein expression

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Polypeptide signal sequences of modified fragments of human immunoglobulin heavy chain binding protein (Bip) are disclosed. Also disclosed are fusion proteins comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide. Also disclosed are protein expression vectors comprising a promoter operably linked to a first DNA sequence encoding a signal sequence comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) and a second DNA sequence encoding a heterologous polypeptide fused in frame to the first DNA sequence. Further disclosed are methods of producing a polypeptide comprising expressing a fusion protein comprising a polypeptide signal sequences of modified fragments of human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide and recovering the heterologous polypeptide.

In eukaryotes, protein synthesis for nearly all proteins begins in the cytoplasm via mega protein complexes called ribosomes. Various proteins complete their synthesis and folding in the cytoplasm and remain there where they function. However, many others are exported out of the cytoplasm and into the endoplasmic reticulum (ER) where they acquire the needed post-translational modifications to attain their proper protein structure and biological activity prior to export to their intended cellular locations or to the cell surface or secreted out of cells. Proteins destined for export out of the cytoplasm are distinguished from cytoplasmic proteins by a specialized protein element at the amino (N-) terminus called the signal sequence.

Signal sequences (also called signal peptides) have no consensus amino acid sequence or length but typically comprise the initial 15-40 residues at the N-terminus with 7-20 contiguous hydrophobic amino acid residues which form an a-helical secondary structure that is often flanked by charged residues. Signal sequences are identified in the cytoplasm by a specialized multi-subunit protein:RNA complex called the signal recognition particle (SRP) which directs these nascent proteins to specialized pores within the ER

membrane called translocons where these proteins are transported across the ER membrane into the ER lumen- a process known as protein translocation.

Protein translocation occurs concurrently during protein synthesis in mammals while in other eukaryotes, this process can be either co-or post-translational. Signal sequence-mediated protein translocation is also utilized in bacteria for directing proteins out of the cytoplasm and into the periplasm. In mammals, signal sequences are identified by SRP as they emerge from ribosomes which temporarily pauses protein translation to allow the targeting of the entire SRP-nascent protein-ribosome complex to translocons via the associated SRP receptor. Protein synthesis is resumed after SRP is released and the ribosome-nascent protein complex is properly docked at the translocon.

Most enzyme and other protein therapeutics are produced by recombinant technology that is designed to secrete these recombinant proteins out of cells and into cell culture to simplify downstream purification. These recombinant enzymes and other proteins therefore must utilize signal sequences and this same cellular pathway for secretion.

High-level production of these proteins therefore requires signal sequences that can mediate efficient ER

targeting and protein translocation across the ER membrane. However, signal sequences are not equivalent for facilitating ER targeting and translocation. The identification of signal sequences by SRP is believed to occur rapidly and efficiently, but the subsequent ER

targeting and translocation steps are highly disparate among proteins. Because signal sequences are recognized twice, first by SRP for targeting the nascent protein-ribosome complex to ER and subsequently by translocon proteins (i.e., Sec61 proteins) and other translocon-associated ER

proteins to initiate translocation, both are potential sites for regulation.

This latter step has been shown to be much more stringent and less efficient and thus, is a major bottleneck in this process. Surprisingly, most signal sequences are intrinsically inefficient for facilitating protein translocation. Consequently, many ER-targeted nascent protein-ribosome complexes dissociate from the ER membrane and protein synthesis is aborted, thereby reducing their protein expression and secretion.

The present invention provides polypeptide signal sequences, comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip).

The present invention also provides fusion proteins, comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide.

Further provided are protein expression vectors, comprising a promoter operably linked to a first DNA sequence wherein the first DNA sequence encodes a polypeptide signal sequence comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence, and a second DNA sequence which is fused in frame to the first DNA sequence, wherein the second DNA sequence encodes a heterologous polypeptide.

The present invention also provides methods for producing a polypeptide, comprising expressing a fusion protein comprising a polypeptide signal sequence derived from human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide and recovering said heterologous polypeptide.

Also disclosed are methods of producing a polypeptide, comprising expressing a fusion protein comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) polypeptide signal sequence operably linked to a heterologous polypeptide, and recovering said heterologous polypeptide.

The present invention further provides protein expression vectors, comprising a promoter operably linked to a first DNA sequence wherein said first DNA sequence encodes a polypeptide signal sequence comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) and a second DNA sequence fused in frame to said first DNA sequence, wherein said second DNA sequence encodes a heterologous polypeptide.

Also disclosed are methods for increasing protein expression, comprising expressing a fusion protein comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) and a heterologous protein, and recovering the heterologous protein.

The disclosed invention also provides for methods of increasing protein secretion, comprising expressing a fusion protein comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) and a heterologous protein, and recovering the heterologous protein.