title: Directed evolution of mammalian anti-apoptosis proteins by somatic hypermutation.
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.
authors.affiliation: Department of Chemical and Biomolecular Engineering,  The Johns Hopkins University, 3400 North Charles Street, 221 Maryland Hall, Baltimore, MD 21218-2694,  USA.
authors.forename: Brian S
authors.initials: BS
authors.lastname: Majors
authors.forename: Gisela G
authors.initials: GG
authors.lastname: Chiang
authors.forename: Nels E
authors.initials: NE
authors.lastname: Pederson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Betenbaugh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D019020:Directed Molecular Evolution / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008322:Mammals / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012845:Sindbis Virus / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D051020:bcl-X Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D051020:bcl-X Protein; D053148:Caspase 3; D019311:Staurosporine