Heterologous Protein Production Using The Twin Arginine Translocation Pathway

Abstract

Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

1 . A method for evaluating and identifying putative substrates utilizing a Tat secretory pathway, comprising comparing know and recorded amino acid sequences of a polypeptide in a microorganism against the Tat signal sequence motif, wherein the Tat signal sequence has the motif XRRXX within a first 35 N′ terminal residues of the amino acid sequence of the polypeptide, wherein RR represents two adjacent arginine residues, and X designates positions restricted to other selected amino acid residues. 
     
     
         2 . The method of  claim 1 , wherein the Tat signal sequence motif (XRRXX) is identified as (X 1 )R 0 R +1  (X +2 )(X +3 )(X +4 ), wherein X −1  is selected from the group consisting of M, H, A, P, K, R, N, T, G, S, D, Q and E; wherein R 0 R +1  represent the twin arginines, wherein X +2  is selected from the group consisting of A, P, K, R, N, T, G, S, D, Q and E; wherein X +3  is selected from the group consisting of I, W, F, L, V, Y, M, C, H, A, P, N and T; and wherein X +4  is selected from the group consisting of A, Q, I, L, V, M and F. 
     
     
         3 . The method of  claim 2 , wherein the Tat signal sequence motif (XRRXX) is identified as (X −1 )R 0 R +1 (X +2 )(X +3 ))(X +4 ), wherein X −1  is selected from the group consisting of M, H, A, P, K, R, N, T, G, S, D, Q and E; wherein R 0 R +1  represent the twin arginines, wherein X +2  is selected from A, P, K, R, N, T, G, S, D, Q and E; wherein X +3  is selected from the group consisting of I, W, F, L, V, Y, M, C, H, A, P, N and T; and wherein X +4  is selected from the group consisting of Q, I, L, V, M and F. 
     
     
         4 . The method of  claim 3 , wherein the Tat signal sequence motif (XRRXX) is identified as (X −1 ) R 0 R +1 (X +2 )(X +3 )(X +4 ), wherein X −1  is selected from the group consisting of H, A, P, K, R, N, T, G, S, D, Q and E; wherein R 0 R +1  represent the twin arginines, wherein X +2  is selected from the group consisting of A, P, K, R, N, T, G, S, D, Q and E; wherein X +3  is selected from the group consisting of I, W, F, L, V, Y, M, C, H, A, P, N and T; and wherein X +4  is selected from the group consisting of Q, I, L, V, M and F. 
     
     
         5 . A method for secreting a heterologous protein from a host microorganism comprising operably ligating a nucleic acid sequence encoding the heterologous protein to a Tat signal sequence selected from those identified in accordance with  claim 2 , and inserting the ligated pair into an expression vector in the host microorganism, expressing the heterologous protein under the control of the Tat signal sequence and secreting the heterologous protein from the microorganism via the Tat expression pathway. 
     
     
         6 . The method of  claim 5 , wherein the expressed heterologous protein is secreted in its correctly-folded, active form. 
     
     
         7 . The heterologous protein that is expressed and secreted from a host microorganism via the Tat expression pathway in accordance with  claim 5 . 
     
     
         8 . The correctly-folded active heterologous protein that is expressed and secreted from a host microorganism via the Tat expression pathway in accordance with  claim 6 . 
     
     
         9 . A method for secreting a heterologous protein from a host microorganism comprising operably ligating a nucleic acid sequence encoding the heterologous protein to a Tat signal sequence selected from those identified in accordance with  claim 3 , and inserting the ligated pair into an expression vector in the host microorganism, expressing the heterologous protein under the control of the Tat signal sequence and secreting the heterologous protein from the microorganism via the Tat expression pathway. 
     
     
         10 . The method of  claim 9 , wherein the expressed heterologous protein is secreted in its correctly-folded, active form. 
     
     
         11 . The heterologous protein that is expressed and secreted from a host microorganism via the Tat expression pathway in accordance with  claim 9 . 
     
     
         12 . The correctly-folded active heterologous protein that is expressed and secreted from a host microorganism via the Tat expression pathway in accordance with  claim 10 . 
     
     
         13 . A method for secreting a heterologous protein from a host microorganism comprising operably ligating a nucleic acid sequence encoding the heterologous protein to a Tat signal sequence selected from those identified in accordance with  claim 4 , and inserting the ligated pair into an expression vector in the host microorganism, expressing the heterologous protein under the control of the Tat signal sequence and secreting the heterologous protein from the microorganism via the Tat expression pathway. 
     
     
         14 . The method of  claim 13 , wherein the expressed heterologous protein is secreted in its correctly-folded, active form. 
     
     
         15 . The heterologous protein that is expressed and secreted from a host microorganism via the Tat expression pathway in accordance with  claim 13 . 
     
     
         16 . The correctly-folded active heterologous protein that is expressed and secreted from a host microorganism via the Tat expression pathway in accordance with  claim 14 .