High level of expression of INGAP in bacterial and eukaryotic cells

Abstract

Removal of the nucleotide sequence encoding the signal peptide from the INGAP coding sequence allows cultured cells to express substantial amounts of INGAP activity. Previous attempts have provided only low yields of INGAP, possibly because the signal sequence of INGAP is toxic to the cells.

1. A recombinant construct for expression of Islet Neogenesis Associated Protein or INGAP activity  a protein which stimulates islet cell neogenesis comprising:
 a first nucleotide sequence encoding amino acids  residues 27 to 175 as shown in SEQ ID NO: 6 operably linked to a transcriptional initiation site and a translational initiation site, wherein a second nucleotide sequence encoding a signal peptide is not present immediately 5′ of said first nucleotide sequence.  
 
     
     
       2. The construct of  claim 1  wherein nucleotides 1-16 of SEQ ID NO: 1 are not present 5′ of said first nucleotide sequence. 
     
     
       3. The construct of  claim 1  further comprising a third nucleotide sequence encoding a histidine tag. 
     
     
       4. The construct of  claim 3  wherein the third nucleotide sequence is immediately 5′ or 3′ to said first nucleotide sequence. 
     
     
       5. The construct of  claim 1  wherein the transcriptional initiation site is inducible. 
     
     
       6. The construct of  claim 1  wherein the transcriptional initiation site is the lac promoter/  and operator. 
     
     
       7. The construct of  claim 1  further comprising a promoter sequence  wherein the transcriptional initiation site is capable of initiating constitutive transcription. 
     
     
       8. The construct of  claim 7  wherein the promoter sequence  transcriptional initiation site is Rous sarcoma virus long terminal repeat (RSVLTR). 
     
     
       9. The construct of  claim 1  further comprising a nucleotide sequence encoding a nuclear antigen. 
     
     
       10. The construct of  claim 9  wherein the nuclear antigen is Epstein-Barr nuclear antigen- 1  (EBNA-1). 
     
     
       11. The construct of  claim 1  further comprising an origin of replication. 
     
     
       12. The construct of  claim 11  wherein the origin of replication is Epstein Bar Virus (EBV) origin of replication. 
     
     
       13. A method of producing biologically active Islet Neogenesis Associated Protein or INGAP protein  from a recombinant host cell comprising the steps of:
 culturing a host cell comprising a recombinant construct comprising a first nucleotide sequence encoding amino acids  residues 27 to 175 as shown in SEQ ID NO: 6 operably linked to a transcriptional initiation site and a translational initiation site, wherein a second nucleotide sequence encoding a signal peptide is not present immediately 5′ of said first nucleotide sequence, and  
 recovering protein from said cultured host cell.  
 
     
     
       14. The method of  claim 13  wherein the construct further comprises a third nucleotide sequence encoding a histidine tag, and INGAP protein  is purified using a nickel affinity matrix. 
     
     
       15. A  An isolated host cell comprising a recombinant construct comprising a first nucleotide sequence encoding amino acids 27 to 175 as shown in SEQ ID NO: 6 operably linked to a transcriptional iron  initiation site and a translational initiation site, wherein a second nucleotide sequence encoding a signal peptide is not present immediately 5+   5 ′ of said first nucleotide sequence. 
     
     
       16. The construct of  claim 1  wherein the first nucleotide sequence encoding amino acids  residues 27 to 175 comprises nucleotides 12-456 of SEQ ID NO: 4. 
     
     
       17. The method of  claim 13  wherein the first nucleotide sequence encoding amino acids  residues 27-175 comprises nucleotides 12-456 of SEQ ID NO: 4. 
     
     
       18. The host cell of  claim 15  wherein the first nucleotide sequence encoding amino acids  residues 27-175 comprises nucleotides 12-456 of SEQ ID NO: 4. 
     
     
       19. The construct of  claim 1  wherein the transcriptional initiation site is selected from the group consisting of: λcI promoter, tac promoter, trp promoter, and tet promoter. 
     
     
       20. The construct of  claim 1  which comprises a nucleotide sequence as shown in SEQ ID NO:  4 . 
     
     
       21. A pair of oligonucleotide primers for amplifying a coding sequence consisting of nucleotides  12  to  456  of SEQ ID NO:  4 , wherein each of said oligonucleotide primers hybridizes to an opposite strand of a double- stranded INGAP template under conditions sufficient for amplifying, wherein a first of said oligonucleotide primers hybridizes to the  5 ′ end of the coding sequence for mature human INGAP and the second of said oligonucleotide primers hybridizes to the  3 ′ end of the nucleotide sequence encoding mature human INGAP under conditions sufficient for amplifying nucleotides  12  to  456  of SEQ ID NO:  4 .   
     
     
       22. The pair of oligonucleotide primers of  claim 21  wherein one primer has the nucleotide sequence shown in SEQ ID NO:  2  and one primer has the nucleotide sequence shown in SEQ ID NO:  3 . 
     
     
       23. A method of making an expression construct for producing INGAP in a recombinant host cell, comprising the step of:
   linking a transcription initiation site, a translation initiation site, and a coding sequence for mature human INGAP consisting of nucleotides  12  to  456  of SEQ ID NO:  4 , to make an expression construct which is devoid of the signal sequence of the coding sequence of INGAP.     
     
     
       24. The method of  claim 23  further comprising linking to said coding sequence for mature human INGAP a coding sequence for a histidine tag. 
     
     
       25. The method of  claim 23  wherein the transcription initiation site is inducible. 
     
     
       26. The method of  claim 25  wherein the transcription initiation site is selected from the group consisting of the lac promoter/operator, the tac promoter, the trp promoter, the λcI promoter, and the tet promoter. 
     
     
       27. The method of  claim 23  wherein the coding sequence for mature human INGAP is obtained by amplification of a coding sequence consisting of nucleotides  12  to  456  of SEQ ID NO:  4 . 
     
     
       28. The method of  claim 27  wherein the amplification is performed using primers having sequences as shown in SEQ ID NO:  2  and SEQ ID NO:  3 . 
     
     
       29. A recombinant construct for expression of a protein which stimulates islet cell neogenesis, comprising:
   a first nucleotide sequence encoding mature human INGAP consisting of nucleotides  12  to  456  of SEQ ID NO:  4 , said first nucleotide sequence being operably linked to a transcriptional initiation site and a translational initiation site, wherein a second nucleotide sequence encoding a signal peptide according to SEQ ID NO:  5  is not present immediately  5 ′ of said first nucleotide sequence.     
     
     
       30. The construct of  claim 29  wherein nucleotides  1 - 16  of SEQ ID NO:  1  are not present  5 ′ of said first nucleotide sequence. 
     
     
       31. The construct of  claim 29  further comprising a third nucleotide sequence encoding a histidine tag. 
     
     
       32. The construct of  claim 29  wherein the third nucleotide sequence is immediately  5 ′ or  3 ′ to said first nucleotide sequence. 
     
     
       33. The construct of  claim 29  wherein the transcriptional initiation site is inducible. 
     
     
       34. The construct of  claim 33  wherein the transcriptional initiation site is the lac promoter/operator. 
     
     
       35. The construct of  claim 29  wherein the transcriptional initiation site is capable of initiating constitutive transcription. 
     
     
       36. The construct of  claim 35  wherein the promoter sequence is Rous sarcoma virus long terminal repeat ( RSVLTR ). 
     
     
       37. The construct of  claim 29  further comprising a nucleotide sequence encoding a nuclear antigen. 
     
     
       38. The construct of  claim 37  wherein the nuclear antigen is Epstein- Barr nuclear antigen -   1   ( EBNA -   1   ). 
     
     
       39. The construct of  claim 29  further comprising an origin of replication. 
     
     
       40. The construct of  claim 39  wherein the origin of replication is Epstein Bar Virus ( EBV )  origin of replication.   
     
     
       41. The construct of  claim 33  wherein the transcriptional initiation site is the λcI promoter/operator. 
     
     
       42. The construct of  claim 33  wherein the transcriptional initiation site is the trp promoter. 
     
     
       43. The construct of  claim 33  wherein the transcriptional initiation site is the tac promoter. 
     
     
       44. The construct of  claim 33  wherein the transcriptional initiation site is the tet promoter. 
     
     
       45. A method of producing biologically active Islet Neogenesis Associated Protein ( INGAP )  from a recombinant host cell comprising the steps of:      culturing a host cell comprising a recombinant construct comprising a first nucleotide sequence encoding mature human INGAP consisting of nucleotides  12  to  456  of SEQ ID NO:  4  operably linked to a transcriptional initiation site and a translational initiation site, wherein a second nucleotide sequence encoding a signal peptide according to SEQ ID NO:  5  is not present immediately  5 ′ of said first nucleotide sequence; and        recovering protein from said cultured host cell.     
     
     
       46. The method of  claim 45  wherein the construct further comprises a third nucleotide sequence encoding a histidine tag, and INGAP is purified using a nickel affinity matrix. 
     
     
       47. A host cell comprising a recombinant construct comprising a first nucleotide sequence encoding mature human INGAP operably linked to a transcriptional initiation site and a translational initiation site, wherein a second nucleotide sequence encoding a signal peptide according to SEQ ID NO:  5  is not present immediately  5 ′ of said first nucleotide sequence. 
     
     
       48. The method of  claim 23  wherein the coding sequence for mature human INGAP encodes amino acid residues  27  to  175  as shown in SEQ ID NO:  6 . 
     
     
       49. The pair of oligonucleotide primers of  claim 21  wherein the first of said oligonucleotide primers comprises nucleotides  12  to  31  of SEQ ID NO:  2  and the second of said oligonucleotide primers comprises nucleotides  13  to  32  of SEQ ID NO:  3 .