Valproic acid: A viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures

Abstract

Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of approximately 2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHO-DG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.

Characterization of a Single b-type Heme, FAD, and Metal Binding Sites in the Transmembrane Domain of Six-transmembrane Epithelial Antigen of the Prostate (STEAP) Family Proteins

“…Furthermore, we found that Steap3 expression was enhanced when valproic acid was added to the transfection medium. Valproic acid is a histone deacetylase inhibitor that has been shown previously to enhance transient gene expression in HEK-293 cells (14). The presence of valproic acid in the 293F transfection medium resulted in a ϳ1.6-fold increase in expression of Steap3-Venus over unsupplemented expression.…”

A Novel Germ Cell-specific Protein, SHIP1, Forms a Complex with Chromatin Remodeling Activity during Spermatogenesis

“…Total RNA Extraction and RT-PCRs-Total RNA samples obtained from seven tissues (testis, brain, liver, spleen, kidney, skeletal muscle, and ovary) of adult mouse, cDNA from germ cell-devoid testes of W/W v mutant mice, and prepubertal and adult male mice (aged 8,10,12,14,16,20,30, and 84 days) were used for reverse transcription. Total RNA was extracted using TRIzol TM Reagent (MRC) according to the manufacturer's protocol, and cDNA was synthesized by random hexamer and oligo(dT) priming with Omniscript reverse transcriptase (Qiagen).…”

“…Protein Samples and Western Blot Analysis-Western blot analysis was performed using protein samples from the testes of prepubertal and adult male mice (aged 8,10,12,14,16,20,30, and 84 days) as well as germ cell-devoid testes of W/W v mutant mice. Testicular cells and sperm from testes of normal adult male mice were isolated by suspension in 52% isotonic Percoll (GE Healthcare) and centrifugation for 10 min (27,000 ϫ g, 10°C) and were resuspended in Mg 2ϩ -Hepes buffer (7).…”

“…The supernatant was removed, and testicular cells in the pellet were washed with Dulbecco's modified Eagle's medium. The isolated testicular cells were cultured using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 20 mM valproic acid (VPA, Sigma) at 37°C with 5% CO 2 for 20 h (8). After 20 h, we observed that somatic cells (Ͻ5%) isolated from mouse testis stuck to the dish, and the isolated testicular cells (Ͼ95%) were still in suspension.…”

“…T, testis; B, brain; Li, liver; S, spleen; K, kidney; SK, skeletal muscle; Ov, ovary; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. B, stage-specific expression of Kctd19 was determined from mouse testes on different days after birth (8,10,12,14,16,20,30, and 84 days). C, total lysates from wild-type (WT) mouse testes and germ cell-lacking testes of W/W v mutant mice were immunoblotted with the anti-KCTD19 antibody.…”