Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B.
Insulin is a polypeptide hormone which is produced by the beta-cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des-threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des-threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2-3 at 25 degrees C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des-threonine insulin and hence increased the production yield of active insulin.
Son
Young-Jin
YJ
Interdisciplinary Program for Bioengineering, Seoul National University, Seoul 151-742, Korea.
Kim
Chang-Kyu
CK
Kim
Young Bong
YB
Kweon
Dae-Hyuk
DH
Park
Yong-Cheol
YC
Seo
Jin-Ho
JH
D000595:Amino Acid Sequence; D043424:Carboxypeptidase B / Q000737:chemistry* / Q000378:metabolism; D002949:Citraconic Anhydrides / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D011384:Proinsulin / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D002949:Citraconic Anhydrides; D011384:Proinsulin; D043424:Carboxypeptidase B; D014357:Trypsin