Single-step trypsin cleavage of a fusion protein to obtain human insulin and its C peptide.
The kinetics for trypsin cleavage of different fusion proteins, consisting of human proinsulin and two IgG-binding domains (ZZ), were investigated. To achieve simultaneous removal of the fusion tag and processing of proinsulin to insulin and free C peptide, three versions of the ZZ-proinsulin fusion protein were generated, having different trypsin-sensitive cleavage sites, Arg, Lys-Arg or Lys. The ZZ-proinsulin fusion proteins which accumulated as inclusion bodies in Escherichia coli cells were solubilized, refolded and purified by IgG affinity chromatography. The yield of ZZ-proinsulin monomers exceeded 90%. The kinetics for the trypsin cleavage revealed unexpected differences when comparing the three linkers and it was found that the single arginine linker was most efficiently processed. Characterization of the cleavage products by reverse-phase chromatography, mass spectrometry and N-terminal sequencing verified that human insulin and C peptide were generated. The results demonstrate that high yields of native insulin, C peptide and affinity tag can be achieved by simultaneous cleavage of a fusion protein at three different trypsin-sensitive sites in a single step. The implications for production and recovery of various recombinant proteins are discussed.
Jonasson
P
P
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Nilsson
J
J
Samuelsson
E
E
Moks
T
T
Ståhl
S
S
Uhlén
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002096:C-Peptide / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013205:Staphylococcal Protein A / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism*
D002096:C-Peptide; D017931:DNA Primers; D007328:Insulin; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D011384:Proinsulin; D014357:Trypsin