Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris.
The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.
Sun
J
J
Department of Medicine, Monash Medical School, Box Hill, Australia.
Coughlin
P
P
Salem
H H
HH
Bird
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015843:Serpins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013917:Thrombin / Q000037:antagonists & inhibitors; D014361:Trypsin Inhibitors / Q000096:biosynthesis
D011994:Recombinant Proteins; D015843:Serpins; D014361:Trypsin Inhibitors; C094472:serpin B6; D013917:Thrombin