Biology Reference
In-Depth Information
2
CHAPTER
Protein Engineering as
an Enabling Tool for
Synthetic Biology
Patrick C. Cirino and Shuai Qian
University of Houston, Houston, TX, USA
INTRODUCTION
Proteins are essential ingredients in any natural or synthetic biological system, and the
extent to which properties of one or more proteins can be modified in direct and desired
ways often dictates success in synthetic biology and metabolically engineered systems.
Common examples of protein properties that may need modification include stability
(e.g. to solvents, low pH, or thermal denaturation), binding affinity, catalytic activity, and
substrate specificity. Enzymes are often engineered to carry out new functions in the context
of synthetic metabolic pathways. As synthetic biologists are often concerned with achieving
novel control over gene expression, regulatory proteins and transcription factors engineered
to have new or improved regulatory properties can also play a powerful role in this
discipline. This chapter describes a variety of the most common and effective methods in
which existing proteins are engineered for improved or even novel properties. Note that a
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(even for the case of
noncatalytic proteins). We first introduce the general concept of protein engineering and the
two broadest categories of this practice
property
may also be referred to as a
fitness
parameter, or
activity
rational design versus directed evolution.
Following a closer look into rational protein design, we then describe a variety of
techniques used by protein engineers to construct protein libraries and to screen these
libraries using high-throughput assays or cell growth-based selections. Finally, we present
several recent and/or pioneering examples in which protein engineering served as an
enabling tool in synthetic biology applications.
The protein engineering methods described here all involve introducing changes at the
DNA level (i.e. altering the corresponding gene sequence). Specified changes to a DNA
sequence originate from DNA synthesis (refer to Chapter 1 for detailed discussion of DNA
synthesis technologies). Short oligonucleotides may be constructed (e.g. PCR primers) and
used to introduce desired mutations using any number of gene amplification, assembly,
or recombination techniques, or alternately the entire gene may be synthesized and directly
cloned into an expression vector. While this chapter does not describe details behind the
molecular biology used to engineer proteins, it is important to note that in all cases
the changes made are at the genetic level and the engineered protein is expressed in an
appropriate host organism. In some cases a single protein scaffold serves as the
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wild-type
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