Protein secretion

Abstract

DNA constructs, host cells and production methods are disclosed for the expression and recovery of polypeptides, especially those altered to have one or more glycosylation sites added or deleted. The DNA constructs, host cells and methods provided herein employ a DNA segment corresponding to a mammalian tissue plasminogen activator signal and/or pro peptide.

What is claimed is:  
     
         1 . A DNA construct comprising: 
 a first DNA segment encoding a precursor polypeptide comprising a prosequence of a mammalian t-PA; and    a second DNA segment operably linked to the first DNA sequence, the second DNA sequence encoding a heterologous glycoprotein.    
     
     
         2 . The DNA construct of  claim 1  wherein the heterologous glycoprotein is an immunoadhesin.  
     
     
         3 . The DNA construct of  claim 2  wherein the immunoadhesin is a TNF receptor immunoadhesin.  
     
     
         4 . The DNA construct of  claim 3  wherein the TNF receptor immunoadhesin is TNFRI-IgG1.  
     
     
         5 . The DNA construct of  claim 1  wherein the DNA encoding the mammalian t-PA pro-sequence is operably linked to a pre-sequence other than a mammalian t-PA pre-sequence.  
     
     
         6 . The DNA construct of  claim 5  wherein the heterologous glycoprotein is and immunoadhesin.  
     
     
         7 . The DNA construct of  claim 6  wherein the immunoadhesin is a TNF receptor immunoadhesin.  
     
     
         8 . The DNA construct of  claim 7  wherein the TNF receptor immunoadhesin is TNFR1-IgG1.  
     
     
         9 . The DNA construct of  claim 5  wherein the mammalian t-PA pro-sequence is operably linked a pre-sequence associated with the native heterologous polypeptide.  
     
     
         10 . The DNA construct of  claim 9  wherein the heterologous glycoprotein is a TNF receptor immunoadhesin and the pre-sequence is a pre-sequence of a mammalian TNF receptor.  
     
     
         11 . The DNA construct of  claim 10  wherein the mammalian t-PA pro-sequence is SEQ ID NO: 7.  
     
     
         12 . The DNA construct of  claim 11  wherein the pre-sequence is SEQ ID NO: 8.  
     
     
         13 . The DNA construct of  claim 12  wherein the TNF receptor immunoadhesin is TNFR1-IgG1.  
     
     
         14 . A DNA construct comprising: 
 a first DNA segment encoding a precursor peptide; and    a second DNA segment operably linked to the first DNA sequence, the second DNA sequence encoding a heterologous glycosylation site variant glycoprotein.    
     
     
         15 . The DNA construct of  claim 14  further comprising one or more additional DNA segments operably linked to the first and second DNA segments.  
     
     
         16 . The DNA construct of  claim 14  wherein the precursor peptide comprises the pro-sequence of a mammalian t-PA.  
     
     
         17 . The DNA construct of  claim 16  wherein the pro-sequence is a human t-PA prosequence.  
     
     
         18 . The DNA construct of  claim 17  wherein the pro-sequence is SEQ ID NO: 5.  
     
     
         19 . The DNA construct of  claim 16  further comprising a pre-sequence of a mammalia t-PA.  
     
     
         20 . The DNA construct of  claim 19  wherein the pre-sequence is a human t-PA pre-sequence.  
     
     
         21 . The DNA construct of  claim 20  wherein the pre-sequence is SEQ ID NO: 3.  
     
     
         22 . The DNA construct of  claim 21  wherein the precursor peptide is SEQ ID NO 1.  
     
     
         23 . The DNA construct of  claim 14  wherein the heterologous glycosylation site variant is a glycosylation site addition variant.  
     
     
         24 . The DNA construct of  claim 14  wherein the heterologous glycosylation site variant is a glycosylation site deletion variant.  
     
     
         25 . The DNA construct of  claim 24  wherein the heterologous glycosylation site variant is an immunoadhesin.  
     
     
         26 . The DNA construct of  claim 25  wherein the immunoadhesin is TNFR-IgG.  
     
     
         27 . The DNA construct of  claim 26  wherein the TNFR-IgG is TNFR1-IgG1.  
     
     
         28 . The DNA construct of  claim 27  wherein the TNFR1-IgG1 has an N-linked glycosylation site selected from the group consisting of amino acid positions 14, 105, 111 and 248 deleted.  
     
     
         29 . The DNA construct of  claim 28  wherein the TNFR1-IgG1 has the N-linked site at 14 deleted.  
     
     
         30 . A cultured eukaryotic host cell comprising a DNA construct comprising: 
 a first DNA segment encoding a precursor peptide corresponding to a mammalian tissue plasminogen activator secretory peptide; and    a second DNA segment operably linked to the first DNA sequence, the second DNA sequence encoding a heterologous glycosylation site variant.    
     
     
         31 . The cultured eukaryotic host cell of  claim 30  wherein the host cell is a rodent host cell.  
     
     
         32 . The cultured eukaryotic host cell of  claim 31  which is a CHO cell.  
     
     
         33 . A method of producing a polypeptide which has been altered to delete one or more native N-linked glycosylation sites comprising the steps of 
 (a) culturing a eukaryotic host cell comprising a DNA construct comprising: 
 a first DNA segment encoding a precursor peptide corresponding to a mammalian tissue plasminogen activator signal-pro peptide; and  
 a second DNA segment operably linked to the first DNA sequence, the second DNA sequence encoding a heterologous glycosylation site deletion variant polypeptide;  
 wherein the eukaryotic host cell express the first and second DNA segments and the polypeptide is secreted from the cell; and  
   (b) recovering the polypeptide so produced.