Page 263 - Advances in Textile Biotechnology

244 Advances in textile biotechnology

These extracts essentially lack both the genomic material and the cellular
boundary system but they contain the cellular components required for
transcription and/or translation of genes. Cell-free translation systems,
provide a useful alternative to the cellular-expression systems as they can
synthesize large proteins with a speed and accuracy that approach those of
in vivo translation and they can express proteins that would otherwise
interfere with host cell physiology such as protein aggregation in inclusion
bodies or proteolysis owing to endogenous proteases which are encoun-
tered with the cellular host. It is also a very useful system for isotope label-

ing of proteins and synthesis of artificial proteins that contain unnatural
amino acids (Sawasaki et al., 2002).

10.5 Purification of recombinant proteins
Unless recombinant proteins are being secreted to the extracellular space,
the high-level gene expression in the cytosolic compartment usually requires
either cell lysis by high-pressure homogenization or sonication or lysis by
freeze–thaw procedures with lysozyme to rescue the protein of interest

(Hallberg, 2008). Most protein purification techniques are based on the
intrinsic differences in the physicochemical properties of proteins, such as
solubility, size, charge, hydrophobicity and shape. Commonly used methods
that exploit these physicochemical properties of proteins include precipita-
tion, dialysis, electrophoresis and chromatography, the latter being, by far,
the most widely used (Chow et al., 2008).
Recent reports have included downstream processing strategies for

recovering recombinant proteins, by the inclusion of affinity tags for puri-

fication purposes. The use of affinity tags enables various proteins to be

purified using conventional chromatographic purification techniques, which

rarely have adverse affects on biological or biochemical activity. Affi nity

tags can be typically defined as exogenous amino acid sequences with a high
affinity for a specific biological or chemical ligand. His-tags are the most

widely used affinity tags and their purification is based on the use of che-
lated metal ions as affinity ligands. Other groups include tags that use

antibody based purification (FLAG, Softag1, Softag3, Streptag II) or anti-
body purification using protein A affinity chromatography. The choice of a

suitable affinity tag depends both on the type of application for the protein
of interest and the stage of development of the protein but it is additionally
conditioned by the cost of the chromatographic support and the scalability
of the process.

Once affinity tags have served for extracting the target protein from the
crude extract, a tag-removal step is usually developed for obtaining the pure

detagged protein. Most of the available methods for affinity tag removal

include enzymatic cleavage of the tag followed by specific removal of the
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