TY - JOUR
T1 - Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin
AU - Mackin, Robert B.
AU - Choquette, Meredith H.
N1 - Funding Information:
The authors thank Dr. Yousef Basir and Eva Lovas (Creighton University) for performing mass spectrometric analysis of the peptides, and Steve Kelly (Creighton University) and Sheila Norton (University of Nebraska Medical Center) for performing DNA sequence analysis. We also thank Dr. FuSheng Shen (laboratory of Dr. Y. Peng Loh, National Institute of Child Health and Human Development) for performing the initial mutation to generate the H10D proinsulin and Eli Lilly and Co. for providing normal human proinsulin. The University of Nebraska Medical Center DNA sequencing core facility was supported by NIH Grant CA 36727, and the Creighton University mass spectrometry core facility was supported by NIH Grant RR 16469. Work in the authors’ laboratory was supported by NIH Grant DK 52085.
PY - 2003/2
Y1 - 2003/2
N2 - Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein's affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1.
AB - Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein's affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1.
UR - https://www.scopus.com/pages/publications/0037296164
UR - https://www.scopus.com/pages/publications/0037296164#tab=citedBy
U2 - 10.1016/S1046-5928(02)00643-5
DO - 10.1016/S1046-5928(02)00643-5
M3 - Article
C2 - 12597879
AN - SCOPUS:0037296164
SN - 1046-5928
VL - 27
SP - 210
EP - 219
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -