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Abstract The cellular environment is dynamic and complex, involving thousands of different macromolecules with total concentrations of hundreds of grams per liter. However, most biochemistry is conducted in dilute buffer where the concentration of macromolecules is less than 10 g/L. High concentrations of macromolecules affect protein stability, function, and protein complex formation, but to understand these phenomena fully we need to know the concentration of the test protein in cells. Here, we quantify the concentration of an overexpressed recombinant protein, a variant of the B1 domain of protein G, in Tuner (DE3)™Escherichia colicells as a function of inducer concentration. We find that the protein expression level is controllable, and expression saturates at over 2 mMupon induction with 0.4 mMisopropyl β‐d‐thiogalactoside. We discuss the results in terms of what can and cannot be learned from in‐cell protein NMR studies inE. coli.
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Dynamic Light Scattering Microrheology of Phase-Separated Poly(vinyl) Alcohol–Phytagel Blends
In this investigation, we explored the microrheological characteristics of dilute hydrogels composed exclusively of Poly(vinyl) alcohol (PVA), Phytagel (PHY), and a blend of the two in varying concentrations. Each of these polymers has established applications in the biomedical field, such as drug delivery and lens drops. This study involved varying the sample concentrations from 0.15% to 0.3% (w/w) to assess how the concentration influenced the observed rheological response. Two probe sizes were employed to examine the impact of the size and verify the continuity hypothesis. The use of two polymer blends revealed their immiscibility and tendency to undergo phase separation, as supported by the existing literature. Exploring the microrheological structure is essential for a comprehensive understanding of the molecular scale. Dynamic light scattering (DLS) was chosen due to its wide frequency range and widespread availability. The selected dilute concentration range was hypothesized to fall within the transition from an ergodic to a non-ergodic medium. Properly identifying the sample’s nature during an analysis—whether it is ergodic or not—is critical, as highlighted in the literature. The obtained results clearly demonstrate an overlap in the results for the storage (G’) and loss moduli (G″) for the different probe particle sizes, confirming the fulfillment of the continuum hypothesis.
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- Award ID(s):
- 2114565
- PAR ID:
- 10580550
- Publisher / Repository:
- MDPI Polymers
- Date Published:
- Journal Name:
- Polymers
- Volume:
- 16
- Issue:
- 20
- ISSN:
- 2073-4360
- Page Range / eLocation ID:
- 2875
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/polym16202875
