| 摘要 |
Disclosed is a method of producing a protein which comprises: (a) producing a protein which comprises a first polypeptide contiguous with an intein, which intein is contiguous with a second polypeptide comprising a binding domain, wherein said first polypeptide is a fusion polypeptide comprising a heterologous N terminal signal sequence which can direct secretion of the protein, wherein protein production comprises transfecting an eukaryotic cell with a nucleic acid, which nucleic acid comprises (i) a first portion which encodes the polypeptide, contiguous with (ii) a second portion which encodes the intein, contiguous with (iii) a third portion which encodes a binding domain, under conditions such that the eukaryotic cell expresses and secretes the protein; (b) generating a thioester intermediate following an intein autocleavage reaction and adding a cysteine, homocysteine, selenocysteine, homoselenocysteine or a functional analogue thereof to the C-terminus by native chemical ligation, wherein, when the eukaryotic cell is a human cell, the protein is produced ex vivo. |
