Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-12-21
2003-11-18
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S007700, C435S069100, C435S320100, C435S252100, C435S183000, C435S822000, C530S350000, C536S023100, C436S501000, C436S519000, C436S536000, C436S063000
Reexamination Certificate
active
06649376
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is phycobiliprotein-assisted expression and folding of proteins and the production of function-added recombinant phycobiliproteins.
BACKGROUND OF THE INVENTION
Many foreign proteins expressed in bacteria form insoluble aggregates called inclusion bodies. Recovery of the recombinant protein of interest requires dissolving the inclusion bodies under denaturing conditions, removing the denaturant to allow the recombinant protein to fold, and finally purifying the recombinant protein. This process is laborious and frequently gives low yields. Inclusion bodies are formed because the aggregation of the newly synthesized recombinant polypeptide is faster than its folding into the native structure. In one aspect, the invention provides bifunctional fusion constructs which are rapidly incorporated into a macromolecular structure such that the fusion proteins are separated from one another and unable to self-associate.
In a more particular aspect, the macromolecular structures are oligomeric phycobiliproteins—a family of structurally related photosynthetic accessory proteins naturally found, inter alia, in cyanobacteria, the chloroplasts of the Rhodophyta (red algae) and in those of the Cryptophyceae. When they carry covalently attached linear tetrapyrrole prosthetic groups (bilins), these proteins can exhibit brilliant colors and intense fluorescence, making them valuable specific labeling reagents. Unfortunately, the diversity of such reagents has been restricted to naturally occurring phycobiliproteins and their target specificity generated by chemical conjugation, which generates heterogeneity (see, e.g. Siiman et al., 1999, Bioconj Chem, 10, 1090-1106). The use of phycobiliproteins in the invention also overcomes these prior art limitations and provides homogenous, specific labeling reagents.
SUMMARY OF THE INVENTION
We have found that phycobiliprotein subunits are tolerant to terminal extensions of different sizes. A phycobiliprotein subunit domain in a fusion protein is able to assemble with a cognate partner subunit to form heterodimers, and frequently further assemble into even higher-order aggregates, and into the light-harvesting antenna complexes, phycobilisomes. The non-phycobiliprotein part of the fusion protein (the displayed domain) is exposed on the surface of the oligomeric phycobiliproteins. We define this fusion protein expression system as “phycobilisome display”. Newly synthesized fusion polypeptides are quickly separated from each other by virtue of the assembly of the phycobiliprotein domain (the carrier domain) into oligomers. The non-phycobiliprotein moiety (the displayed domain) of the fusion protein is displayed on the surface of oligomer, e.g. the rods of the phycobilisome, and is able to fold into functional proteins while sequestered from interaction with other unfolded polypeptides. Phycobilisome display can therefore be used as an alternative folding environment for difficult-to-fold proteins, especially those that have been found difficult to fold in other organisms. The displayed protein can be used as a fusion construct bearing a fluorescent phycobiliprotein tag, or be separated from the carrier phycobiliprotein domain by cleavage of the linker peptide between the carrier domain and the displayed domain by a specific protease. Either embodiment may be practiced in cells (e.g. using a resident protease) or in vitro (e.g. using purified fusion proteins, cell-free expression systems, etc.), though cleavage is preferably practiced outside the cell to control its timing with respect to folding.
Accordingly, the invention provides methods and compositions for displaying a functional polypeptide domain on an oligomeric phycobiliprotein. The compositions include fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein. In particular embodiments, the phycobiliprotein domain is a natural phycobiliprotein domain or modified variant thereof; the functional oligomeric phycobiliprotein is an &agr;, &bgr; heterodimer; the displayed domain comprises an affinity tag, an oligomerization moiety, a specific binding moiety and/or a signaling moiety; and/or the displayed domain is refractive to expression in
E. coli
. The compositions also include functional oligomeric phycobiliproteins comprising the subject fusion proteins and cells comprising such oligomeric phycobiliproteins.
The subject methods include methods for making a functional displayed domain comprising the step of combining a polypeptide comprising a displayed domain and a phycobiliprotein domain with a phycobiliprotein subunit under conditions to form a subject fusion protein. In particular embodiments, the methods further comprise prior to the combining step, the step of making the polypeptide by expressing a nucleic acid encoding the polypeptide; and/or after the combining step, the step of separating the functional displayed domain from the functional phycobiliprotein domain. The methods steps may occur intracellularly, e.g. in a cell which is or is a progeny of a natural cell which naturally makes functional phycobiliprotein, or a cell engineered to produce functional phycobiliprotein.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation.
The invention provides methods and compositions for displaying a functional polypeptide (the foreign protein or displayed domain) on an oligomeric phycobiliprotein. The compositions include fusion proteins comprising a functional displayed domain and a functional phycobiliprotein domain incorporated in a functional oligomeric phycobiliprotein.
A functional phycobiliprotein domain is capable of assembling the fusion protein in a functional oligomeric phycobiliprotein. Preferred domains provide at least 1%, preferably at least 10%, more preferably at least 50%, more preferably at least 75%, more preferably at least 90% and most preferably substantially equivalent oligomer assembly ability as that of a corresponding unfused phycobiliprotein, as measured in competition assays, e.g. as described herein.
Any phycobiliprotein domain having the requisite functionality may be used and these may be derived from natural, semisynthetic or synthetic sequences. A wide variety of natural phycobiliproteins are known in the art, e.g. Apt and Grossman, 1995, J Mol Biol 248, 79-96, including proteins derived from many cyanobacteria, rhodophytes (red algae) and cryptomonads, etc. (see, e.g. Glazer, 1994, J Appl Phycol 6, 105-112; Glazer et al. 1995, Photosynth Res 46, 93-105), particularly phycoerythrins, phycocyanins, and allophycocyanins. In addition, a wide variety of methods are known for modifying such natural sequences to generate semi-synthetics, e.g. Glazer, 1994, supra, describes phycobiliproteins having non-natural, predetermined bilin compositions, and Toole et al., 1998, Mol Micro 30, 475-486 describes recombinations of phycobiliprotein deletion mutants. Finally, known phycobiliprotein structure-function relationships (e.g. Anderson et al., 1998, Mol Micro 30, 467-474) are exploited to generate synthetic sequence analogs using conventional methods.
In a particular embodiment, the phycobiliprotein domain is characterizable as an &agr; or &bgr; subunit, based on its sequence similarity to natural &agr; and &bgr; phycobiliprotein subunits. The selection of &agr; or &bgr; phycobiliprotein domains may yield different results, affecting the displayed domain, the carrier protein, or both, and such differences guide the selection of the carrier phycobiliprotein subunit. For example, the phycocyanin &agr; subunit and the &bgr;-L11-&agr; subunit-fusion are preferred when the foreign protein is displayed on the N-terminus of the carrier protein because of the higher yield and the better spectroscopic properties of the resulting fusion protein. For display on the C-terminus, phycocyanin &bgr; subunit and
Cai Yuping
Glazer Alexander N.
Kam Chih-Min
Low Christopher S. F.
Osman Richard Aron
The Regents of the University of California
LandOfFree
Multifunctional recombinant phycobiliprotein-based... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Multifunctional recombinant phycobiliprotein-based..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Multifunctional recombinant phycobiliprotein-based... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3184805