Directed evolution of mammalian anti-apoptosis proteins by somatic hypermutation

Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, 221 Maryland Hall, Baltimore, MD 21218-2694, USA.
Protein Engineering Design and Selection (Impact Factor: 2.54). 12/2011; 25(1):27-38. DOI: 10.1093/protein/gzr052
Source: PubMed

ABSTRACT Recently, researchers have created novel fluorescent proteins by harnessing the somatic hypermutation ability of B cells. In this study, we examined if this approach could be used to evolve a non-fluorescent protein, namely the anti-apoptosis protein Bcl-x(L), using the Ramos B-cell line. After demonstrating that Ramos cells were capable of mutating a heterologous bcl-x(L) transgene, the cells were exposed to multiple rounds of the chemical apoptosis inducer staurosporine followed by rounds of recovery in fresh medium. The engineered B cells expressing Bcl-x(L) exhibited progressively lower increases in apoptosis activation as measured by caspase-3 activity after successive rounds of selective pressure with staurosporine treatment. Within the B-cell genome, a number of mutated bcl-x(L) transgene variants were identified after three rounds of evolution, including a mutation of Bcl-x(L) Asp29 to either Asn or His, in 8 out of 23 evaluated constructs that represented at least five distinct Ramos subpopulations. Subsequently, Chinese hamster ovary (CHO) cells engineered to overexpress the Bcl-x(L) Asp29Asn variant showed enhanced apoptosis resistance against an orthogonal apoptosis insult, Sindbis virus infection, when compared with cells expressing the wild-type Bcl-x(L) protein. These findings provide, to our knowledge, the first demonstration of evolution of a recombinant mammalian protein in a mammalian expression system.

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    • "One approach to increase yields from mammalian bioprocesses is to increase the viable cell number by reducing the rate of apoptosis. Therefore, multiple cell engineering strategies were developed to increase apoptosis resistance of CHO cells by overexpression of endogenous (Han et al., 2011) or evolved anti-apoptotic proteins of the Bcl-family (Majors et al., 2012). Sophisticated transcriptomic, proteomic and metabolomic approaches identified bottlenecks in the energy metabolism of CHO cells that prevent efficient growth and/or protein production (Chong et al., 2010; Doolan et al., 2010). "
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