Biology Reference
In-Depth Information
of flow cytometry as one of the most enabling tools in protein engineering and synthetic
biology. Below we describe some key applications of FACS for protein engineering.
Furthermore, the
section of this chapter describes the use of FACS for
engineering effector specificity of a regulatory protein.
Cell Surface Display If a protein
'
Applications
'
s fluorescence-linked property can be measured
intracellularly, then FACS isolation of the corresponding cell and genetic information is
straightforward. However, if the protein does not fold or function properly intracellularly,
or if the protein
'
s substrate, ligand, or function-linked fluorophore do not readily cross the
cell membrane, then the assay must take place extracellularly, presenting the challenge
of linking genotype to phenotype. For FACS applications, a variety of cell surface display
methods have been developed and implemented for bacteria, yeast, insect, and
mammalian cells. 55 The general approach is to recruit the host
'
s native machinery for
protein translocation and membrane anchoring by fusing the protein of interest to a
'
'
carrier protein
'
system.
In one example, Georgiou and coworkers used E. coli surface display and multiparameter flow
cytometry to engineer a native outer membrane protease with altered substrate specificity. 56
In this system, the peptide substrate containing a fluorophore and FRET quenching partner
separated by the target scissile bond coat the E. coli surface displaying the protease variants.
Cleavage at the target peptide disrupts the FRET interaction, resulting in fluorescence. To
include specificity in the screen, an alternate fluorescent counterselection substrate containing
an undesired protease cleavage site was also used. This approach resulted in a protease variant
having high specificity and activity on a nonnative substrate.
A wide variety of proteins can be displayed on the yeast surface. 55 While cloning and
transformation efficiencies are often lower in yeast compared to bacteria, yeast
surface display is often preferred due to its native eukaryotic secretory pathway. Yeast surface
display is a proven system for selecting antibodies with improved affinity, specificity, and
stability. Wittrup and coworkers developed the Aga1-Aga2 display system in Saccharomyces
cerevisiae . 57 The Aga1 protein is naturally covalently attached to the cell wall and forms
disulfide bonds with Aga2. Fusing the protein of interest to Aga2 results in surface display.
Epitope tags can be included in the protein fusions to allow immunofluorescent
quantification of displayed protein and normalization of activity or binding. This system
has been widely used for affinity maturation of antibody
33
antigen interactions, as well as
protein interactions. 55,58
In Vitro Compartmentalization An alternate approach to using cells as expression hosts is
to express protein libraries in vitro. 59 In order to maintain a link between genotype and
phenotype, however, compartmentalization is needed. In this approach, microscopic
emulsion compartments are generated as artificial cells. The gene library is transcribed and
translated within the emulsion droplets, so as long as an assay can be developed in which
droplet fluorescence correlates with protein function, FACS can be used for sorting while
maintaining the genotype
other protein
phenotype linkage. Reaction conditions or solubilities that may
be incompatible with in vivo systems may be more compatible with emulsions, and
limitations associated with transport across cell membranes are eliminated. This technique
was first reported for the selection of a phosphotriesterase from 3.4
10 7 library members
with 63-fold improved activity relative to wild-type. 60 Water-in-oil (w/o) emulsions with
10 10 droplets per ml of emulsion can be created, with the majority containing a single
variant, enabling screening of 10 8
3
10 11 variants. 61
Similarly, emulsions can also be used for compartmentalizing whole cells, e.g. if the
fluorescence signal would otherwise diffuse away from the cell. In the direction evolution of
serum paraoxonase (PON1), a library of variants was expressed in E. coli , and single cells were
encapsulated in w/o/w emulsion droplets. 59 Droplets with improved thiolactonase activity
were isolated by FACS using a fluorogenic thiol-detecting dye that is retained in the droplets.
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