Light-activated reassembly of split green fluorescent protein.

Truncated green fluorescent protein (GFP) with the 11th ß-strand removed is potentially interesting for bioconjugation, imaging, and the preparation of semisynthetic proteins with novel spectroscopic or functional properties. Surprisingly, the truncated GFP generated by removing the 11th strand, once refolded, does not reassemble with a synthetic peptide corresponding to strand 11 but does reassemble following light activation. The mechanism of this process has been studied in detail by absorption, fluorescence, and Raman spectroscopy. The chromophore in this refolded truncated GFP is found to be in the trans configuration. Upon exposure to light a photostationary state is formed between the trans and cis conformations of the chromophore, and only truncated GFP with the cis configuration of the chromophore binds the peptide. A kinetic model describing the light-activated reassembly of this split GFP is discussed. This unique light-driven reassembly is potentially useful for controlling protein-protein interactions.

Synthetic control of green fluorescent protein.

Semisynthetic green fluorescent proteins (GFPs) can be prepared by producing truncated GFPs recombinantly and assembling them with synthetic beta-strands of GFP. The yield from expressing the truncated GFPs is low, and the chromophore is either partially formed or not formed. An alternative method is presented in which full-length proteins are produced recombinantly with a protease site inserted between the structural element to be removed and the rest of the protein. The native peptide can then be replaced by cutting the protease site with trypsin, denaturing in guanidine hydrochloride to disrupt the complex, separating the native peptide from the rest of the protein by size exclusion, and refolding the protein in the presence of a synthetic peptide. We show that this method allows for removal and replacement of the interior chromophore containing helix and that the GFP barrel is capable of inducing chromophore formation in a synthetic interior helix.

Deconstructing green fluorescent protein.

Green fluorescent protein (GFP) has been reassembled from two pieces, a large fragment 214 amino acids in length that is produced recombinantly (GFP 1-10) and a short synthetic peptide corresponding to the 11th stave of the beta-barrel that is 16 amino acids long (synthetic GFP 11), following a system developed by Waldo and co-workers (Cabantous, S.; et al. Nat. Biotechnol. 2005, 23, 102-7) as an in vivo probe for protein association and folding. We demonstrate that the reassembled protein has identical absorption and excited-state proton transfer dynamics as a whole protein of the identical sequence. We show that the reassembled protein can be taken apart and the peptide replaced with a different synthetic peptide designed to perturb the chromophore absorption. Thus, this semisynthetic reassembly process offers a general route for studying the assembly of the beta-barrel as well as the introduction of unnatural amino acids.

Solution pH jump during antibody and Fc-fusion protein thaw leads to increased aggregation.

Freeze-thaw cycles impact the amount of aggregation observed in antibodies and Fc-fusion proteins. Various formulation strategies are used to mitigate the amount of aggregation that occurs upon putting a protein solution through a freeze-thaw cycle. Additionally, low pH solutions cause native antibodies to unfold, which are prone to aggregate upon pH neutralization. There is great interest in the mechanism that causes therapeutic proteins to aggregate since aggregate species can cause unwanted immunogenicity in patients. Herein, an increase in aggregation is reported when the pH is adjusted from pH 3 up to a pH ranging from pH 4 to pH 7 during the thaw process of a frozen antibody solution. Raising the pH during the thaw process caused a significant increase in the percent aggregation observed. Two antibodies and one Fc-fusion protein were evaluated during the pH jump thaw process and similar effects were observed. The results provide a new tool to study the kinetics of therapeutic protein aggregation upon pH increase.

Therapeutic ultrasound: Increased HDL-Cholesterol following infusions of acoustic microspheres and apolipoprotein A-I plasmids.

BACKGROUND: Low levels of HDL-C are an independent cardiovascular risk factor associated with increased premature cardiovascular death. However, HDL-C therapies historically have been limited by issues relating to immunogenicity, hepatotoxicity and scalability, and have been ineffective in clinical trials. OBJECTIVE: We examined the feasibility of using injectable acoustic microspheres to locally deliver human ApoA-I DNA plasmids in a pre-clinical model and quantify increased production of HDL-C in vivo. METHODS: Our novel site-specific gene delivery system was examined in naïve rat model and comprised the following steps: (1) intravenous co-administration of a solution containing acoustically active microspheres (Optison™, GE Healthcare, Princeton, New Jersey) and human ApoA-I plasmids; (2) ultrasound verification of the presence of the microspheres within the liver vasculature; (3) External application of locally-directed acoustic energy, (4) induction of microsphere disruption and in situ sonoporation; (4) ApoA-I plasmid hepatic uptake; (5) transcription and expression of human ApoA-I protein; and (6) elevation of serum HDL-C. RESULTS: Co-administration of ApoA-I plasmids and acoustic microspheres, activated by external ultrasound energy, resulted in transcription and production of human ApoA-I protein and elevated serum HDL-C in rats (up to 61%; p-value < 0.05). CONCLUSIONS: HDL-C was increased in rats following ultrasound directed delivery of human ApoA-I plasmids by microsphere sonoporation. The present method provides a novel approach to promote ApoA-I synthesis and nascent HDL-C elevation, potentially permitting the use of a minimally-invasive ultrasound-based, gene delivery system for treating individuals with low HDL-C.