Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence.
We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.
Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
L R
LR
Castellanos-Serra
E
E
Hardy
R
R
Ubieta
N S
NS
Vispo
C
C
Fernandez
V
V
Besada
V
V
Falcon
M
M
Gonzalez
A
A
Santos
G
G
Perez
A
A
Silva
L
L
Herrera
D000595:Amino Acid Sequence; D002096:C-Peptide / Q000378:metabolism*; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007328:Insulin / Q000378:metabolism*; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics*; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011384:Proinsulin / Q000737:chemistry / Q000235:genetics*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002096:C-Peptide; D007328:Insulin; D007376:Interleukin-2; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011384:Proinsulin; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin; D008239:Lysine