Recombinant carrier molecule for expression, delivery and purification of target polypeptides

Abstract

Recombinant carrier molecules having amino acid sequences from thermostable enzymes and methods of use for expression, recovery and delivery of foreign sequences (peptides and polypeptides) produced in different systems (bacteria, yeast, DNA, cell cultures such as mammalian, plant, insect cell cultures, protoplast and whole plants in vitro or in vivo are provided. The recombinant carrier molecule using sequences from lichenase B (Lic B) were also made and used it as part of carrier protein to express, recover and deliver a variety of target polypeptides of interest.

1 . A recombinant carrier molecule, comprising: 
 (a) a modified mature polypeptide of a thermostable enzyme lacking one or more amino acid segments, wherein the modified mature polypeptide retains its thermostability; or    (b) a substantially complete mature polypeptide of the thermostable enzyme suitable for fusing to a heterologous polypeptide at each of N-terminus and C-terminus of the mature polypeptide, and optionally in the loop region.    
     
     
         2 . The recombinant carrier molecule of  claim 1 , wherein the mature polypeptide of (a) is modified in that it lacks a loop region or has a disrupted loop region, or has at least one unique restriction site in the loop region not naturally present therein.  
     
     
         3 . The recombinant carrier molecule of  claim 2 , wherein the thermostable enzyme is lichenase B.  
     
     
         4 . The recombinant carrier molecule of  claim 3 , wherein the modified mature polypeptide or the substantially complete mature polypeptide has a disrupted loop region.  
     
     
         5 . The recombinant carrier molecule of  claim 4 , wherein the mature polypeptide has two identifiable regions delineated by the loop region, wherein the orientation or location of the two identifiable regions are different from that in the wild-type mature polypeptide.  
     
     
         6 . A carrier protein comprising a recombinant carrier molecule fused to at least one heterologous polypeptide, wherein the recombinant carrier molecule has 
 (a) a modified mature polypeptide of the thermostable enzyme lacking one or more amino acid segments, wherein the modified mature polypeptide retains its enymatic activity or thermostability or    (b) a complete or substantially complete mature polypeptide of the thermostable enzyme is suitable for carrying a heterologous polypeptide fused at each of N-terminus and C-terminus of the mature polypeptide, and optionally in the loop region.    
     
     
         7 . The carrier protein of  claim 6 , wherein the heterologous polypeptide is fused to the recombinant carrier molecule at at least one fusion site selected from the group consisting of the N-terminus, the C-terminus and an internal fusion site.  
     
     
         8 . The carrier protein of  claim 6 , wherein the heterologous polypeptide is fused to the carrier molecule at an internal fusion site which is located at a region that links the two domains of the mature polypeptide.  
     
     
         9 . A carrier protein comprises 
 a recombinant carrier molecule having sequences from lichenase B protein; and    one or more heterologous polypeptides linked to the carrier molecule as N terminal, C-terminal or internal fusions,    wherein the carrier protein is thermostable.    
     
     
         10 . The carrier protein of  claim 9 , wherein the carrier molecule is encoded by a sequence set forth in SEQ ID NO: 1 or 2.  
     
     
         11 . The carrier protein of  claim 9 , wherein the recombinant carrier molecule is about 28 kD.  
     
     
         12 . A method for the production of a carrier protein in a plant comprising: 
 (a) providing a plant containing an expression cassette having a nucleotide sequence encoding a carrier protein comprising a recombinant carrier molecule having amino acid sequences from a mature thermostable polypeptide, wherein the nucleotide sequence is operably linked such that expression of the cassette results in translation of the carrier protein;    (b) growing said plant under conditions whereby a nucleotide sequence is expressed and the carrier protein is produced; and    (c) optionally recovering the carrier protein.    
     
     
         13 . The method of  claim 12 , wherein the promoter is selected form the group consisting of plant constitutive and plant tissue specific promoters.  
     
     
         14 . The method of  claim 12 , wherein the carrier protein is expressed in leaf root or seed of the plant.  
     
     
         15 . The method of  claim 12 , wherein the plant is a dicot or a monocot.  
     
     
         16 . The method of  claim 12 , wherein the amino acid sequences from a mature thermostable polypeptide are sufficient to confer thermostability to the carrier protein.  
     
     
         17 . A method for recovering a polypeptide from prokaryotic or eukaryotic cells after expression inside said cells of a carrier protein, wherein the carrier protein comprises a recombinant carrier molecule derived from Lie B protein; and one or more heterologous polypeptides linked to the carrier molecule as N-terminal, C-terminal or internal fusions, the method comprising: 
 (a) lysing said cells;    (b) heating said lysate sufficient to denature non-thermostable proteins;    (c) separating soluble material from insoluble material after step (b) by a separation device, and    (d) recovering the carrier protein in the soluble material after step (c).    
     
     
         18 . The method of  claim 17 , wherein the separation device is a centrifugation device.  
     
     
         19 . The method of  claim 17 , wherein the recombinant carrier molecule is fused to two or more contiguous or non-contiguous epitope-containing segments.  
     
     
         20 . The method of  claim 19 , wherein the epitope-containing segments being fused to the recombinant carrier molecule at fusion sites selected from the group consisting of the N-terminus, C-terminus and an internal fusion site.  
     
     
         21 . The method of  claim 20 , wherein the recombinant carrier molecule is encoded by a nucleic acid sequence set forth in SEQ ID NO: 1 or 2.