Cytochalasin D can improve heterologous protein productivity in adherent Chinese hamster ovary cells.
We generated a series of adherent gene-amplified CHO clones expressing human secreted alkaline phosphatase (SEAP) as a model for heterologous protein production. Clones demonstrate a 26- to 52-fold increase in productivity compared to controls after dhfr/methotrexate-mediated gene amplification and clone selection. SEAP is stably expressed in these clones over at least a 6-week period without significant productivity loss. Two-dimensional protein electrophoresis identified 21 proteins that exhibited altered expression in clones of increasing SEAP productivity. Based on MALDI TOF/TOF mass spectrometry of relevant protein spots, changes in translation, energy pathways, chaperones, regulatory proteins, and cytoskeletal proteins were observed, including a 4-fold expression increase in actin capping protein. We hypothesized that an alteration of the actin cytoskeleton using cytochalasin D as a mimic for actin-capping protein could have a beneficial effect on heterologous protein secretion. Treatment with 0.5 mug/mL cytochalasin D increased SEAP productivity 2- to 3-fold compared to an amplified control which resulted in an increase in productivity from 52- to 150-fold compared to a nonamplified parent.
Hayduk
Eric J
EJ
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, USA.
Lee
Kelvin H
KH
D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D015638:Cytochalasin D / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; D015638:Cytochalasin D