Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells.
Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships.
Emmons
Thomas L
TL
Pfizer, Inc., Global Research and Development, St. Louis Laboratories, 700 Chesterfield Parkway West, Chesterfield, MO 63017-1732, USA.
Mathis
Karl J
KJ
Shuck
Mary E
ME
Reitz
Beverly A
BA
Curran
Daniel F
DF
Walker
Mark C
MC
Leone
Joseph W
JW
Day
Jacqueline E
JE
Bienkowski
Michael J
MJ
Fischer
H David
HD
Tomasselli
Alfredo G
AG
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000502:physiology; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000166:cytology* / Q000821:virology*; D007700:Kinetics; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry; D000071756:Soluble Guanylyl Cyclase
D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D012685:Sepharose; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase