PROLink-Single Step Circularization and Purification Procedure for the Generation of an Improved Variant of Human Growth Hormone.
Human growth hormone (hGH) plays an important role during human development and is also an approved therapeutic for the treatment of several diseases. However, one major drawback of hGH is its short circulating half-life requiring frequent administration, which is inconvenient and painful for the patients. Recent publications indicate that circularization greatly increases the stability of proteins due to their protection from exoproteolytic attack and a higher thermal stability of the circular form. Using sortase A, a transpeptidase isolated from Staphylococcus aureus, we developed a single step solid-phase circularization and purification procedure resulting in a circular version of hGH with improved properties. We could show that circular hGH binds to the recombinant hGH receptor with binding kinetics similar to those of linear hGH and that circularization does not alter the biological activity of hGH in vitro. Besides, circular hGH showed almost complete resistance toward exoproteolytic attack and slightly increased thermal stability which could possibly translate into an extended plasma half-life. The single step solid-phase circularization and purification procedure is in principle a generic process, which could also be applied for other proteins that meet the requirements for circularization.
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt , 64287 Darmstadt, Germany.|Merck KGaA , Frankfurterstrasse 250, 64293 Darmstadt, Germany.
Nicolas
N
Rasche
Merck KGaA , Frankfurterstrasse 250, 64293 Darmstadt, Germany.
Jason
J
Tonillo
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt , 64287 Darmstadt, Germany.|Merck KGaA , Frankfurterstrasse 250, 64293 Darmstadt, Germany.
Marcel
M
Rieker
Merck KGaA , Frankfurterstrasse 250, 64293 Darmstadt, Germany.
Stefan
S
Becker
Brent
B
Dorr
Department of Molecular and Cellular Biology, Harvard University , Cambridge, Massachusetts 02138, United States.|Wyss Institute for Biologically Inspired Engineering , Boston, Massachusetts 02115, United States.
Dmitry
D
Ter-Ovanesyan
Merck KGaA , Frankfurterstrasse 250, 64293 Darmstadt, Germany.
Ulrich A K
UA
Betz
Merck KGaA , Frankfurterstrasse 250, 64293 Darmstadt, Germany.
Björn
B
Hock
Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt , 64287 Darmstadt, Germany.
Harald
H
Kolmar
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D019382:Human Growth Hormone / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013211:Staphylococcus aureus / Q000201:enzymology
D001426:Bacterial Proteins; D019382:Human Growth Hormone; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases