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PHA Granule-Associated Proteins and Their Diverse Functions
Published in Martin Koller, The Handbook of Polyhydroxyalkanoates, 2020
Mariela P. Mezzina, Daniela S. Alvarez, M. Julia Pettinari
The affinity of PhaP for PHA granules has also been used to purify recombinant proteins through simple methods that enable separation of the polymer granules from the cell medium and cellular components or to fuse antigens to PHA nanoparticles. The main phasin from C. necator has been used to construct fusions to several different proteins in PHB-synthesizing E. coli, allowing easy purification of the granule-bound recombinant protein after cell lysis. One study proposed the use of an intein to achieve a self-cleaving fusion, thus enabling the recovery of the protein of interest devoid of PhaP [75]. Polypeptide or protein-ligand fusions to the same phasin were also used as a means to direct PHA nanoparticles to several kinds of human target cells by receptor recognition both in vitro and in an in vivo mouse model [76] and to construct PHA particles that display PhaP-fused antigens that can be used to bind antibodies to enhance fluorescence-activated cell sorting [77]. A different phasin, PhaF from P. putida, was used in similar applications. Proteins fused to the N-terminal domain of this phasin, named BioF, could be produced in P. putida and co-purified with PHA granules, maintaining their enzymatic activity after release [78]. An interesting variant of this system involves the utilization of the PHA granules as substrates for the immobilization of the fused protein, as exemplified by the use of the granule-bound Cry toxin for environmental insect control [79].
Functional Nano-Bioconjugates for Targeted Cellular Uptake and Specific Nanoparticle–Protein Interactions
Published in Grunwald Peter, Biocatalysis and Nanotechnology, 2017
Sanjay Mathur, Shaista Ilyas, Laura Wortmann, Jasleen Kaur, Isabel Gessner
Kim et al., (2010) reported the production of bio-luminescent nano-sensors for the detection of protease activity based on the Au-luciferase conjugate. In this work the alkyne-modified 5 nm Au NPs were attached to azide-modified recombinant luciferase (Luc8-pep-N3) via a Cu(I) catalyzed [3+2] cycloaddition reaction. The click reaction offered an efficient conjugation between NPs and the luciferase protein. The intein-mediated ligation enabled this protein to be site-specifically conjugated on the Au NP. In UV-vis analysis, the final product (Luc8-pep-Au NPs) displayed a slightly red-shifted spectrum (520 nm) because of their surface modification compared to unconjugated NPs (Au-alkyne) with a strong absorption at 516 nm. Bradford assay showed that conjugation offered 4.7 proteins per Au NPs by displacing its monolayer with 2-mercaptoethanol and quantifying the luciferase concentration. The results indicated that the site-specific orientation of luciferase on NPs is critical for detecting protease activity.
Designing an ELP-intein system: toward a more realistic outlook
Published in Preparative Biochemistry and Biotechnology, 2019
Saeed Ranjbar, Fatemeh Rahbarizadeh, Davoud Ahmadvand
Once a tagged protein expressed in an appropriate host cell is recovered from the native proteins of the host, the tag is routinely cleaved off by protease treatment. Tag removal has always been the Achilles’ heel of affinity tagging and one of the common obstacles in the production of recombinant proteins, comprising roughly of over 75% of the downstream purification expenses.[12] In addition to being expensive and including the risk of unspecific cleavage, proteolytic enzymes as the conventional solution create further impurity, which entails additional chromatographic steps to purify the target protein from the cleavage mixture. Hence, the removal of fusion partners adds another layer of complexity and expense to the recombinant protein production process.[13] Intein-mediated protein purification is a self-cleaving method, which applies the intrinsic ability of inteins to self-cleave the purification tag with high accuracy, following a certain chemical change to purify recombinant proteins in a simple and inexpensive approach. The chemical change mostly involves the addition of a thiol such as 1,4-dithiothreitol (DTT), slight shift in pH or an increase in temperature. Expressed as a part of the precursor protein, intein cleaves itself, which results in two new proteins. Modified inteins, explicitly N- or C-terminal cleaving variants, have been introduced to apply in protein purification.[14] When fused to a purification tag, these proteins can give rise to a method for simple tag removal through the addition of a cleaving buffer to the expressed fusion protein solution.[15] We designed and expressed an ELP-intein-tagged GFP, but along the way, we confronted certain complications we deemed necessary to share with potential researchers aiming to work on a similar case.