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


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-xL, using the Ramos B-cell line. After demonstrating that Ramos cells were capable of mutating a heterologous bcl-xL 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-xL 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-xL transgene variants were identified after three rounds of evolution, including a mutation of Bcl-xL 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-xL Asp29Asn variant showed enhanced apoptosis resistance against an orthogonal apoptosis insult, Sindbis virus infection, when
compared with cells expressing the wild-type Bcl-xL 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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    ABSTRACT: A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.
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