Biology Reference
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desirable to use the selection as a preliminary screen, even for the case of random mutation
or DNA shuffling libraries. For larger libraries containing
3 amino substitutions per
variant, or when it is desired to have a high probability of observing all members of a large
library, the selection is an essential first step, though likely to unintentionally eliminate
positive hits, furnish false positives, and provide no kinetic data.
.
Acceptable screening accuracy depends on the functional quality of the library. For example,
random point mutations are not likely to result in drastic improvements in function. The
screen must be sensitive enough to detect low levels of activity/function (particularly in
initial rounds of screening), and also accurate enough to reliably identify variants with
relatively small improvements in function (e.g. 30% improved). There are no steadfast rules
for choosing a mutagenesis strategy or screening system, and a desired level of library
coverage or oversampling is case-dependent. While not discussed here, a variety of research
papers and reviews address statistics and probability relating to protein library construction
and evaluation. 49 51 These can help guide experimental design and interpretation of
screening results and library quality.
High-Throughput Screening in Microtiter Plates
The use of microtiter plate assays is a conventional and straightforward high-throughput
approach to protein library screening. The requirement is that the protein property of
interest can be directly or indirectly measured in the microtiter plate, most commonly
via spectrophotometry or fluorometry. Enzymes have most commonly been engineered
using this approach, since spectroscopic methods are excellent for monitoring rates of
substrate consumption or product formation. Often the assay will employ a surrogate,
colorimetric/fluorometric substrate that is similar in structure/chemistry to the intended
substrate (but includes a chromophore/fluorophore). Linking protein function to
expression of a fluorescent protein is also common. A typical procedure is as follows: a
gene library is constructed and transformed into the expression host of interest. Individual
colonies (representing individual clones) are picked and used to inoculate cultures in
microtiter plate wells. The cultures are replicated for storage and retrieval. Appropriate
growth conditions are used for protein expression and ultimately each culture,
supernatant, or cell lysate, is transferred to a microtiter assay plate to measure optical
properties linked to protein function. Cultures and assays may be carried out in 96-, 384-,
or even 1536-well plates. Colony picking and pipetting robotics are commonly used.
Often a great deal of work goes into assay development, and while this approach is
relatively time-consuming and low-throughput, it can provide much functional detail for
each variant in the library.
32
Flow Cytometry (Fluorescence-Activated Cell Sorting)
Flow cytometry is a technique that allows for the rapid measurement of multiple optical
properties of individual cells, particles, or compartmentalized droplets. Fluorescence-
activated cell sorting (FACS) involves the use of flow cytometry to separate members of
a cell population having specified fluorescent properties (and is not limited to only cells).
Individual FACS instruments are now capable of sorting more than 20 000 cells per
second. The use of FACS for protein engineering requires the generation of a fluorescence
signal that reflects the protein
s property of interest. In the case of engineering proteins
for controlling gene expression, this is as simple as incorporating expression of a
fluorescent protein into the assay. For the case of engineering a protein
'
s ligand-binding
properties, a variety of fluorescent conjugates can be used. The application becomes more
complicated for enzyme engineering, but clever methods have been developed to
accomplish this, as summarized in many reviews. 52,53 An example is the use of a
fluorescence sensor for intracellular pH shifts (
'
), which could be triggered by
hydrolytic enzyme reactions. 54 The high-throughput nature of FACS has led to the use
'
pHluorin
'
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