Process for continuous optimized protein production in insect larvae

Abstract

The present invention provides for a recombinant insect larvae and a process of manufacturing proteins utilizing insect larvae that allows for the selection of individual larvae for harvest at the point of their optimal expression of a protein of interest. This invention also provides for a process to manufacture proteins in larvae that does not require synchronization of the infection, growth and harvest larvae to optimally manufacture a protein of interest. The invention further provides for a process of producing interleukin-2 in larvae.

1. A continuous process for the production of a protein of interest in insect larvae, comprising the steps of:
 a) providing an infected insect larvae food which comprises adding a baculovirus vector to insect larvae food, wherein said baculovirus vector comprising a DNA encoding the protein of interest operatively linked to a gene encoding a green fluorescent protein (GFP) or a GFP mutant, thereby providing a composition of infected insect larvae food;  
 b) continuously infecting insect larvae by feeding said larvae the infected insect larvae food of step (a), thereby resulting in a population of infected insect larvae;  
 c) cultivating the infected insect larvae population of step (b) under suitable conditions for the expression of the protein of interest and the GFP or the GFP mutant by the infected insect larvae population;  
 d) monitoring the infected insect larvae population for the expression of the protein of interest and the GFP or the GFP mutant by measuring the intensity of the fluorescence of GFP or the GFP mutant;  
 e) correlating the intensity of the fluorescence of GFP or the GFP mutant with the amount of the protein of interest expressed;  
 f) harvesting the larvae which express the protein of interest from the population of infected insect larvae;  
 g) recovering the protein of interest from the harvested larvae.  
 
     
     
       2. The process of  claim 1 , wherein said GFP is a GFPuv which has spectral properties that enable quantitation of the protein of interest using fluorescence. 
     
     
       3. The process of  claim 1 , wherein the protein of interest is expressed as a fusion protein with the GFP or the GFP mutant. 
     
     
       4. The process of  claim 1 , wherein step (a) comprises adding 500 μl baculovirus vector loading to insect larvae food, wherein said baculovirus vector loading comprises at least 5×10 7  pfu/ml. 
     
     
       5. The process of  claim 1 , wherein the insect larvae are of the species Trichoplusia ni. 
     
     
       6. The process of  claim 5 , wherein the insect larvae are in the fourth instar. 
     
     
       7. The process of  claim 1 , wherein the DNA encoding the protein of interest and the GFP or the GFP mutant comprise a nucleic acid sequence which encodes an amino acid sequence, wherein said amino acid sequence is cleavable by a specific enzyme which allows the expressed protein of interest to be cleaved off from the GFP or the GFP mutant. 
     
     
       8. The process of  claim 7 , wherein the specific enzyme is enterokinase. 
     
     
       9. The process of  claim 8 , wherein the GFP or the GFP mutant comprises a His-affinity tail. 
     
     
       10. The process of  claim 9 , wherein the protein of interest is interleukin-2. 
     
     
       11. The process of  claim 1 , wherein the infected insect larvae expressing the protein of interest are harvested at least 3-5 days post infection. 
     
     
       12. The process of  claim 1 , wherein the infected insect larvae expressing the protein of interest are harvested at least four days post infection. 
     
     
       13. The process of  claim 1 , wherein the harvested larvae are preserved at a pH of about 3.5 to 4.0. 
     
     
       14. The process of  claim 13 , wherein the harvested larvae are preserved in the presence of protease inhibitors selected from the group consisting of EDTA, PMSF, benzamidine and mixtures thereof. 
     
     
       15. A continuous process for the production of interleukin-2 in insect larvae, comprising the steps of:
 a) providing infected insect larvae food which comprises adding a baculovirus vector to insect larvae food, wherein said baculovirus vector comprises a DNA encoding interleukin-2 operatively linked to a gene encoding a green fluorescent protein (GFP) or a GFP mutant, thereby providing a composition of infected insect larvae food;  
 b) continuously infecting insect larvae by feeding said larvae the infected insect larvae food of step (a), thereby resulting in a population of infected insect larvae;  
 c) cultivating the infected insect larvae population of step (b) under suitable conditions for the expression of interleukin-2 and the GFP or the GFP mutant by the infected insect larvae population;  
 d) monitoring the infected insect larvae population for the expression of interleukin-2 and the GFP or the GFP mutant by measuring the intensity of the fluorescence of GFP or the GFP mutant;  
 e) correlating the intensity of the fluorescence of GFP or the GFP mutant with the amount of interleukin-2 expressed;  
 f) harvesting the larvae which express interleukin-2 from the population of infected insect larvae;  
 g) recovering interleukin-2 from the harvested larvae.  
 
     
     
       16. The process of  claim 15 , wherein the infected insect larvae expressing interleukin-2 are harvested at least 3-5 days post infection. 
     
     
       17. The process of  claim 15 , wherein the harvested larvae are preserved in the presence of protease inhibitors selected from the group consisting of EDTA, PMSF, benzamidine and mixtures thereof. 
     
     
       18. A process for producing a protein of interest from insect larvae, comprising the steps of:
   infecting insect larvae during their growth, with a baculovirus vector comprising a DNA encoding the protein of interest operatively linked to a gene encoding a green fluorescent protein  ( GFP )  or a GFP mutant, so that the protein of interest is expressed as a fusion protein with the GFP or GFP mutant during said growth, wherein the baculovirus vector is associated with the food of the insect larvae to infect the insect larvae;        measuring fluorescence of the GFP or GFP mutant to quantitate the protein of interest in vivo; and        harvesting the insect larvae based on the measured fluorescence, wherein the step of harvesting is continuous; and      
         wherein yield of the protein of interest is increased, in relation to a corresponding process wherein the baculovirus vector does not include said gene encoding a green fluorescent protein  ( GFP )  or a GFP mutant.   
     
     
       19. The process according to  claim 18 , wherein the insect larvae are of the species Trichoplusia ni. 
     
     
       20. The process according to  claim 18 , wherein the protein of interest is interleukin-   2 .   
     
     
       21. The process according to  claim 18 , wherein the GFP or GFP mutant comprises a His- affinity tail.   
     
     
       22. A semi- continuous process for producing a protein of interest from insect larvae, comprising the steps of:      infecting insect larvae during their growth, with a baculovirus vector comprising a DNA encoding the protein of interest operatively linked to a gene encoding a green fluorescent protein  ( GFP )  or a GFP mutant, so that the protein of interest is expressed as a fusion protein with the GFP or GFP mutant during said growth, wherein the baculovirus vector is associated with food of the insect larvae and the insect larvae are infected simultaneously;        measuring fluorescence of the GFP or GFP mutant to quantitate the protein of interest in vivo;        continuously harvesting the insect larvae based on the measured fluorescence due to the expression of the green fluorescent protein  ( GFP )  or the GFP mutant; and        harvesting the protein of interest from the harvested insect larvae,      
         wherein yield of the protein of interest is increased, in relation to a corresponding process wherein the baculovirus vector does not include said gene encoding a green fluorescent protein  ( GFP )  or a GFP mutant.   
     
     
       23. The process according to  claim 22 , wherein the insect larvae are of the species Trichoplusia ni. 
     
     
       24. The process according to  claim 22 , wherein the protein of interest is interleukin-   2 .   
     
     
       25. The process according to  claim 22 , wherein the GFP or GFP mutant comprises a His- affinity tail.