The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus.
Compounds capable of stimulating soluble guanylate cyclase (sGC) activity might become important new tools to treat hypertension. While rational design of these drugs would be aided by elucidation of the sGC three-dimensional structure and molecular mechanism of activation, such efforts also require quantities of high quality enzyme that are challenging to produce. We implemented the titerless infected-cells preservation and scale-up (TIPS) methodology to express the heterodimeric sGC. In the TIPS method, small-scale insect cell cultures were first incubated with a recombinant baculovirus which replicated in the cells. The baculovirus-infected insect cells (BIIC) were harvested and frozen prior to cell lysis and the subsequent escape of the newly replicated virus into the culture supernatant. Thawed BIIC stocks were ultimately used for subsequent scale up. As little as 1 mL of BIIC was needed to infect a 100-L insect cell culture, in contrast to the usual 1L of high-titer, virus stock supernatants. The TIPS method eliminates the need and protracted time for titering virus supernatants, and provides stable, concentrated storage of recombinant baculovirus in the form of infected cells. The latter is particularly advantageous for virus stocks which are unstable, such as those for sGC, and provides a highly efficient alternative for baculovirus storage and expression. The TIPS process enabled efficient scale up to 100-L batches, each producing about 200mg of active sGC. Careful adjustment of expression culture conditions over the course of several 100-L runs provided uniform starting titers, specific activity, and composition of contaminating proteins that facilitated development of a process that reproducibly yielded highly active, purified sGC.
Wasilko
David J
DJ
Pfizer Inc., Global Research and Development Groton/New London Laboratories, Eastern Point Road, Groton, CT 06340, USA.
Lee
S Edward
SE
Stutzman-Engwall
Kim J
KJ
Reitz
Beverly A
BA
Emmons
Thomas L
TL
Mathis
Karl J
KJ
Bienkowski
Michael J
MJ
Tomasselli
Alfredo G
AG
Fischer
H David
HD
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000071756:Soluble Guanylyl Cyclase; D018411:Spodoptera / Q000166:cytology* / Q000378:metabolism* / Q000821:virology; D013997:Time Factors
D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase