Page 264 - Advances in Textile Biotechnology

Developments in recombinant silk and other elastic protein fi bers 245

process enzyme(s) by affinity chromatography. Only in a few specifi c cases

can the affinity tag be removed by harsh chemical treatment (Arnau et al.,

2006). Immobilized metal affinity chromatography (IMAC) is the most

widely used technique for single-step purification of recombinant proteins

containing specific amino acid side chains, particularly those of histidine,
cysteine, and tryptophan as fusion tags from bacterial cell lysates (Bolanos-
Garcia and Davies, 2006).
Novel fusion tags are being developed for increasing solubility such as
NuscA and T7 protein kinase or a small chaperone, Skp; and for aid in
protein folding such as the ubiquitin-based tag SUMO. Others, such as
inteins, are self-cleavable proteases by using either a thiol reagent or pH
and temperature shift to yield intein cleavage and elution of the target
protein (Arnau et al., 2006).
Elastin-like polymers (ELPs) can be used as fusion proteins for easy
purification by two strategies, either engineering the ELP tag into a recom-

binant protein (direct ELP tagging) or recombinantly expressing or chem-

ically attaching to a capture partner that binds specifically to the target
protein in solution (ELP-mediated affi nity capture). The ELP imparts its
phase transition behavior to the target protein and this can be purifi ed by
inverse transition cycling (ITC). This strategy has several advantages over
conventional chromatography making it a universal method for soluble
recombinant proteins; the ELP acts as a capture mechanism, so no chroma-
tography should be needed and the method does not require a concentra-
tion step to recover the final product. In the second approach, ELP-mediated

affinity capture does not require enzymatic or chemical cleavage or the ELP

tag (Chow et al., 2008).
10.6 Experimental characterization of
recombinant proteins
Depending on the individual applications of the obtained biomaterials, and
in order to fully determine the functionality after protein purifi cation when
necessary, several characterization techniques may be required. Many of
them, originally developed for the characterization of proteins, are also
useful for the biophysical characterization of repetitive peptides.
Biophysical characterization can include: the determination of protein
concentration (spectrophotometrically or colorimetrically); the molecular
weight of recombinant proteins can be measured by electrophoresis-based
methods such as SDS-PAGE, mass spectrometry methods such as matrix-
assisted laser desorption ionization (MALDI-MS) or electrospray mass
spectrometry or analytical ultracentrifugation based on sediment coeffi -
cients. Surface plasmon resonance spectroscopy (SPR) provides a way to
determine the binding behavior of peptide-based biomaterials, and binding

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