A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures

Bruno Figueroa, T. M. Sauerwald, G. A. Oyler, J. Marie Hardwick, Michael J. Betenbaugh

Research output: Contribution to journalArticlepeer-review

41 Scopus citations


The overexpression of bcl-2 and its homologues is a widely used strategy to inhibit apoptosis in mammalian cell culture systems. In this study, we have evaluated the Bcl-2 homologue, Bcl-xL and compared its effectiveness to a BCl-XL mutant lacking most of the non-conserved unstructured loop domain, Bcl-XLΔ (deletion of amino acids 26 through 83). The cell line, Chinese hamster ovary (CHO), was genetically modified to express constitutively BCl-XL or the BCl-XL variant and subjected to model apoptotic insults including Sindbis virus (SV) infection, gradual serum withdrawal, and serum deprivation. When cells were engineered to overexpress Bcl-xLΔ, cell death due to the SV was inhibited, and Bcl-xLΔ provided comparable protection to the wild-type BCl-XL even though expression levels were much lower for the mutant. Furthermore, the cells expressing Bcl-xLΔ continued to proliferate following infection while CHO-bcl-xL ceased proliferation immediately following infection. As a result, total production of a heterologous protein encoded on the SV was highest in cell lines expressing Bcl-xLΔ. Cells expressing the variant Bcl-xL also continued to proliferate and showed increased viable cell numbers following gradual serum withdrawal. In contrast, wild-type BCl-XL expressing CHO cells were found to arrest growth but maintain viability following serum withdrawal. Interestingly, CHO cells expressing Bcl-XLΔ were also able to recover and return to rapid growth rates much faster than either the wild-type CHO-bcl-xL or CHO following the replenishment of fresh complete medium containing 10% FBS. Confocal imaging of yellow fluorescent protein (YFP) fused to the N terminus of BCl-XL and Bcl-X LΔ indicated dense aggregates of the Bcl-XLΔ while the wild-type protein was distributed throughout the cell in a manner resembling transmembrane localization. As an alternative to complete removal of the loop domain, BCl-XL variants were created in which aspartate residues containing potential caspase recognition sites within the loop domain of Bcl-xL were removed. Cell populations expressing various Bcl-XL-Asp mutants were exposed to an apoptotic spent medium stimulus, and the cells expressing these Bcl-xL variants provided increased viabilities as compared to cells containing wild-type BCl-X L protein. These studies indicate that modification of anti-apoptotic genes can affect multiple cellular properties including response to apoptotic stimuli and cell growth. This knowledge can be valuable in the design of improved apoptosis inhibitors for biotechnology applications.

Original languageEnglish (US)
Pages (from-to)230-245
Number of pages16
JournalMetabolic Engineering
Issue number4
StatePublished - Oct 2003


  • Apoptosis
  • Bcl-2 family
  • Bcl-x
  • CHO
  • Caspases
  • Chinese Hamster Ovary cells
  • Mammalian cell culture
  • Serum deprivation
  • Sindbis virus infection
  • Spent medium

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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