Identification of anchor points for chemical modification of a small cysteine-rich protein by using a cysteine scan

TY - JOUR

T1 - Identification of anchor points for chemical modification of a small cysteine-rich protein by using a cysteine scan

AU - Vinther, Tine N.

AU - Ribel, Ulla

AU - Pedersen, Thomas Åskov

AU - Kjeldsen, Thomas B.

AU - Jensen, Knud Jørgen

AU - Hubálek, Frantisek

PY - 2011/11/4

Y1 - 2011/11/4

N2 - Chemical modifications of proteins are increasingly important in the development of protein drugs with fine-tuned properties. Regioselective modification, such as the chemoselective alkylation of an unpaired cysteine residue, is a prerequisite for obtaining homogenous protein products. The introduction of an unpaired Cys into the Cys-rich protein, insulin, was investigated by using a Cys scan. This was challenging as the introduced Cys could interfere with insulin's three existing disulfide bonds. However, eight insulin precursors were expressed in Saccharomyces cerevisiae with good yields. Although extensive post-translational modifications of the unpaired Cys were observed, the majority could be removed by selective reduction. An example Cys7 insulin analogue was modified with a PEGylated maleimide moiety. The new variant was active in in vitro and in vivo models. Our results show that even small Cys-rich proteins can be expressed with additional unpaired Cys in meaningful yields and further chemically modified, while maintaining their biological activity.

AB - Chemical modifications of proteins are increasingly important in the development of protein drugs with fine-tuned properties. Regioselective modification, such as the chemoselective alkylation of an unpaired cysteine residue, is a prerequisite for obtaining homogenous protein products. The introduction of an unpaired Cys into the Cys-rich protein, insulin, was investigated by using a Cys scan. This was challenging as the introduced Cys could interfere with insulin's three existing disulfide bonds. However, eight insulin precursors were expressed in Saccharomyces cerevisiae with good yields. Although extensive post-translational modifications of the unpaired Cys were observed, the majority could be removed by selective reduction. An example Cys7 insulin analogue was modified with a PEGylated maleimide moiety. The new variant was active in in vitro and in vivo models. Our results show that even small Cys-rich proteins can be expressed with additional unpaired Cys in meaningful yields and further chemically modified, while maintaining their biological activity.

U2 - 10.1002/cbic.201100464

DO - 10.1002/cbic.201100464

M3 - Journal article

SN - 1439-4227

VL - 12

SP - 2448

EP - 2455

JO - ChemBioChem

JF - ChemBioChem

IS - 16

ER -