Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor.
Therapeutic application of the serpin C1-inhibitor (C1-Inh) in inflammatory diseases like sepsis, acute myocardial infarction and vascular leakage syndrome seems promising, but large doses may be required. Therefore, a high-yield recombinant expression system for C1-Inh is very interesting. Earlier attempts to produce high levels of C1-Inh resulted in predominantly inactive C1-Inh. We describe the high yield expression of rhC1-Inh in Pichia pastoris, with 180 mg/l active C1-Inh at maximum. On average, 30 mg/l of 80-100% active C1-Inh was obtained. Progress curves were used to study the interaction with C1s, kallikrein, coagulation factor XIIa and XIa, and demonstrated that rhC1-Inh had the same inhibitory capacity as plasma C1-Inh. Structural integrity, as monitored via heat stability, was comparable despite differences in extent and nature of glycosylation. We conclude that the P. pastoris system is capable of high-level production of functionally and structurally intact human C1 inhibitor.
Bos
Ineke G A
IG
Dept. of Immunopathology, Sanquin Research at CLB, and Landsteiner Laboratory, Academical Medical Centre, University of Amsterdam, The Netherlands. I.Bos@sanquin.nl
de Bruin
Eric C
EC
Karuntu
Yani A
YA
Modderman
Piet W
PW
Eldering
Eric
E
Hack
C Erik
CE
D003001:Cloning, Molecular; D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D030781:Organisms, Genetically Modified; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D015843:Serpins / Q000096:biosynthesis* / Q000235:genetics
D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D020033:Protein Isoforms; D011994:Recombinant Proteins; C469952:SERPING1 protein, human; D015843:Serpins