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 secreting a heterologous protein from a host microorganism comprising:
 operably ligating a nucleic acid sequence encoding the heterologous protein to a Tat signal sequence motif (XRRXX), 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;   inserting the ligation product 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.   
     
     
         2 . The method of  claim 1 , wherein the expressed heterologous protein is secreted in its correctly-folded, active form. 
     
     
         3 . The heterologous protein, as expressed and secreted from the host microorganism of  claim 1 , via the Tat expression pathway. 
     
     
         4 . The correctly-folded active heterologous protein, as expressed and secreted from a host microorganism of  claim 2 , via the Tat expression pathway.