Library of translational fusion partners for producing recombinant proteins and translational fusion partners screened therefrom

摘要

The invention relates to techniques for the rapid screening of suitable translational fusion partners (TFPs) capable of inducing secretory production of recombinant proteins, especially proteins that are difficult to produce using conventional recombinant production methods.

主权项

1. A method of identifying a target protein specific TFP library, said method comprising:
(i) co-transforming a yeast cell that does not show activity of a reporter protein with a linear vector and a polynucleotide comprising a nucleic acid sequence encoding a target protein to produce a plurality of co-transformed yeast cells, wherein said linear vector comprises a polynucleotide fragment from a library of polynucleotide fragments and a first nucleotide sequence which encodes a portion of the reporter protein comprising a N-terminal amino acid deletion sufficient to substantially eliminate activity of the reporter protein, wherein said polynucleotide comprising said nucleic acid sequence encoding the target protein further comprises, at the 3′ end, a second nucleotide sequence which encodes a portion of the reporter protein comprising the N-terminal amino acids deleted from said reporter protein in said linear vector, and at the 5′ end, a linker DNA; and wherein said target protein is a protein that is difficult to be recombinantly produced by the yeast cell or be secreted therefrom; (ii) incubating said co-transformed yeast cells under conditions effective to allow in vivo recombination between said linear vector and said polynucleotide comprising said nucleic acid encoding the target protein, thereby producing a fusion protein comprising the reporter protein, polynucleotide fragment from a library of polynucleotide fragments and target protein; (iii) identifying the co-transformed yeast cell of (ii) showing an activity of the reporter protein, wherein the reporter protein is secreted as a part of the fusion protein; (iv) identifying the polynucleotide fragment which induces the secretion of said fusion protein as a TFP from the co-transformed yeast cell identified in (iii), wherein the TFP identified in (iv) is a signal sequence.