. 2014 Oct 3;54(16):4011–4020. doi: 10.1021/ie503060a

Table 1. Summary of HTSOS Methods.

methods	screening/library size	advantages	disadvantages	applications
high throughput screening methods
microtiter plates	<10⁴/day	applicable to many assays	laborious	enzyme reactions leading to change in color, fluorescence, pH, cell growth, etc.
DI	limited by transformation efficiency	high sensitivity	not generally applicable	restricted to colorimetric activity assays
FACS	up to 3 × 10⁴/s²³	high sensitivity and extremely high throughput	target enzyme activity has to be coupled with the expression level of fluorescent proteins	enzyme reactions leading to change in fluorescence
cell surface display	limited by transformation efficiency	avoids possible cell lysis during enzyme reaction	protein expressed as fusion proteins	screening for bond-forming enzymes
IVTC	limited by the throughput of the detection method (e.g., FACS) but not transformation efficiency	high sensitivity and efficiency	lack of posttranslational modifications; not suitable for screening enzymes with incompatible conditions between transcription-translation and screening	screening enzymatic activities combined with FACS
RET	limited by transformation efficiency	high sensitivity and efficiency	target enzyme activity has to be coupled with energy transfer between two fluorophores	screening protease activity
high throughput selection methods
plasmid display	limited by transformation efficiency	avoids potential problems of protein secretion, in vitro translation, and/or RNA instability	protein expressed as fusion proteins	selecting protein binders
SNAP display	limited to 10⁹/(1 mL of emulsion)⁵²	resolves proteins with different binding affinities; display multiple copies of a protein; selection under harsh conditions	weak binders can be lost in SNAP monomer display; lack of posttranslational modifications	selecting protein binders
phage display	limited by transformation efficiency	multiple copies of proteins can be displayed	not applicable to selecting diverse enzyme properties; not suitable for selecting eukaryotic proteins; protein expressed as fusion proteins	extremely efficient in affinity-based selections
retrovirus display	limited by transformation efficiency	proper folding of eukaryotic proteins; allows posttranslational modifications	protein expressed as fusion proteins	selecting eukaryotic proteins
mRNA display and ribosome display	not limited by transformation efficiency	overcome the incompatible conditions between in vitro transcription and translation	mRNA instability; lack of posttranslational modifications; protein expressed as fusion proteins	selecting protein binders, high affinity antibodies, and catalytic enzymes
growth complementation	limited by transformation efficiency	potential to select enzymes with diverse properties	selection method needs to be individualized	selecting enzyme properties that can be coupled to host fitness
reporter-based selection	limited by transformation efficiency	potential to select enzymes with diverse properties	reporter needs to be constructed individually according to properties of selected enzyme	selecting enzymes that produce transcription regulation molecules
IVTC	not limited by transformation efficiency	high sensitivity toward variants without target enzyme activity	lack of posttranslational modifications; further screening is needed for identification variants with high enzyme activity⁷⁵	restricted to selecting enzymes that act on DNA