AU2020102633A4 - Oxidation resistant red fluorescent proteins and use thereof - Google Patents

Abstract

We disclosed a red fluorescent protein R3, which is oxidation resistant and oligomer-formation resistant, constructed by removing its cysteine residues. The R3 protein 5 is highly soluble and displays intense red color and fluorescence. We also disclosed a fusion protein of the R3 protein joined by a nanobody as an example of coloring a bioactive protein. The stable red fluorescent protein, as a tracer molecule, will be a valuable tool in location and quantitation of bioactive proteins.

Description

Oxidation resistant red fluorescent proteins and use thereof

Technical field

The field of this invention is biological technology, and more specifically, chromoproteins and fluorescent proteins.

Background

Proteins are usually colorless, because the twenty natural amino acid residues do not absorb visible light appreciably under normal circumstance. Most of the absorbance comes from aromatic residues, such as tryptophan, histidine, phenylalanine, and tyrosine, and proteins with high number of aromatic residues can show their presence when detected in UV region near 280 nm. Fluorescent proteins are a special class of chromoproteins and fluorescent proteins exist in nature. The chromophore is formed by chemical reactions of amino acid residues, such as Ser65-Tyr66-Gly67 in avGFP, due to their spatial proximity and oxidation. The distinct color and fluorescence of these unique proteins has been widely used in biological applications as tracing signals (Cubitt, A. B., R. Heim, S. R. Adams, A. E. Boyd, L. A. Gross and R. Y. Tsien (1995). "Understanding, improving and using green fluorescent proteins." Trends Biochem Sci 20(11): 448-455.). The gene of a fluorescent protein can be easily fused to another gene to produce a fusion protein, which can be expressed inside cells to trace the function of individual proteins or produced in large quality in pure form as biological reagents to study structure and function of proteins, protein-protein interactions, and a range of other biological properties. Fluorescent proteins have cysteines, and their number and locations are unusually not conserved among proteins from different species. Cysteines are chemically active, and they can be oxidized, or react with another cysteine to form a covalent disulfide bond. This chemical activity can cause at least two problems: inactivation by oxidation and oligomer-formation. Oxidation of cysteine residue will cause structural change, leading to change in structure integrity, light absorbance, and fluorescence emission. Formation of unnatural and unintended disulfide bonds results in oligomers, complicating analysis, protein purification, and protein stability. These oxidation and oligomer-formation problems are particularly troublesome when the fluorescent protein or its fusion protein will be used in multiple cellular compartments, oxidative cellular environments, and biological membranes. (Costantini, L. M., M. Baloban, M. L. Markwardt, M. Rizzo, F. Guo, V. V. Verkhusha and E. L. Snapp (2015). "A palette of fluorescent proteins optimized for diverse cellular environments." Nat Commun 6: 7670.). Replacement of cysteine in fluorescent proteins have been attempted, and successful examples exist. The replacement includes Ser, Ala, Val, Met, among others. However, cysteines are usually buried inside a hydrophobic interior, and outcome of the replacement is not predictable, ranging from decreased brightness with single cysteine mutation to complete loss of fluorescence.

Most fluorescent proteins emit green fluorescence, which is noticeable on the dark background. Genetic mutations around the chromophore have expanded the excitation and emission wavelengths to yellow and red region, to created fluorescent proteins cover entire visible spectral range and beyond. This is significant because dark colors, such as red, are more noticeable on a light background. We intend to produce cysteine-free red fluorescent proteins that have intense color and fluorescence and be oxidation resistant and oligomer-formation resistant, to make it stable and easy to use on a light background.

Summary

The object of the present disclosure is to provide a red fluorescent protein that is oxidation resistant and oligomer-formation resistant, due to its lack of cysteines. Itself or its fusion protein can be used as a tracer for location and quantitation. In the first aspect of the present disclosure, it provides the method of making the red fluorescent protein by replacing the cysteine or cystines with an amino acid residue, selected from 20 natural amino acids minus cysteine. In the second aspect of the present disclosure, it provides the method of making the red fluorescent protein, based on a known red fluorescent protein, its sequence set forth by SEQ ID NO: 1, by replacing the cysteine or cystines with an amino acid residue, preferably from the group of amino acids serine, alanine, valine, and methionine. In the third aspect of the present disclosure, it provides the method of making the red fluorescent protein, based on the known red fluorescent protein, its sequence set forth by SEQ ID NO: 1, by replacing its cysteine-30 and cysteine-176 with two valines. The newly created red fluorescent protein R3 contains: (a) the sequence set forth by SEQ ID NO: 2; or (b) a sequence with identity of 90% or more to SEQ ID NO: 2. The fourth aspect of the present disclosure provides a fusion protein, and said fusion protein comprises: (a) the red fluorescent protein according to the first aspect, the second aspect, or the third aspect of the present disclosure; and (b) a fusion part selected from the group comprising antibody, enzyme, transporter, structural protein, and transcription factor. In another preferred embodiment, said antibody is selected from the group comprising nanobody (single domain antibody), antibody, antibody scFv fragment, antibody Fab fragment, and antibody Fc fragment. In another preferred embodiment, said enzyme is selected from the group comprising protease, phosphatase, and kinase. In another preferred embodiment, said fusion part is a protein that has affinity towards another protein target on cell surface or inside cell. In another embodiment, said fusion protein has the red fluorescent protein and the fusion part linked together by a short linker sequence consisting of glycine, serine, or combinations of the two. In another embodiment, said fusion protein has the red fluorescent protein first and the fusion part second, or in reverse order. In another preferred embodiment, said fusion protein contains multivalent (such as bivalent) the red fluorescent protein and the fusion part. The fifth aspect of the present disclosure provides an expression system to produce the red fluorescent protein or the fusion protein comprising the steps of: (a) constructing an expression system for said proteins under a condition suitable for producing the proteins, thereby obtaining a culture containing said proteins; and (b) isolating or recovering said proteins from said culture. In another preferred embodiment, said expression system includes prokaryote and eukaryote. In another preferred embodiment, said expression is carried out in E coli cells or yeast cells. The sixth aspect of the present disclosure provides uses of the red fluorescent protein according to the first, the second, or the third aspect of the present disclosure; or the fusion protein according to the fourth aspect of the present disclosure. In another preferred embodiment, said uses comprise: (a) coloring or labeling the fusion part for visualization to facilitate its purification, localization, and detection; (b) quantitating proteins in western blot, ELISA assay, and other bioassays; (c) bioimaging cellular proteins under microscope or other devices; (d) incorporating into an assay kit for detection and quantitation; (e) revealing abundance and distribution of a target protein in live animal or human for diagnostics. These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and claims.

Description of the drawings

Figure 1. This drawing is to illustrate the selection of proper residues to replace cysteines. Sequence alignment between the fluorescent proteins mCherry and mRuby3 showes the homology between the two proteins. The identical residues in the two proteins are shaded, and the two cysteines (C30 and C176) are highlighted by underline and in bold. This alignment provides the rationale for the equivalent locations of C30 in mRuby3 at 144 in mCherry and C176 in mRuby3 at A180 in mCherry. Figure 2. This drawing is to illustrate the locations of two replaced cysteines (C30V, C176V) in the R3 protein in 3-D space, based on the X-ray structure of mCherry (2h5q). Both C30 and C176 are located on the beta-sheets with their sidechains pointing inwards of the beta barrel. The inwards orientation makes its selection of replacement residues more critical to avoid changes in its structural integrity, because inwards orientation, in comparison with outwards one, will have less space to accommodate the replacement residues. This inwards orientation also suggests the vulnerability of inactivation by oxidation of cysteines, emphasizing the need to make it oxidation resistant. Figure 3. This drawing is to disclose the protein sequence of R3 protein, with a his-tag at its C-terminus, used in our expression system. A skilled person can keep the R3 protein core sequence and add additional sequences or tags for its expression. Figure 4. This drawing is to show the actual purification process of the R3 protein on nickel column. Due to its intense red color, protein movement on the column is clearly visible, following the time sequence of 1-4, indicated by arrows. Figure 5. This drawing is to show the size and purity of the purified red fluorescent protein R3 and the purified fusion protein R3C, as analyzed by PAGE analysis. The R3 protein is shown on the far-right lane (lane 2, 27 kDa), and the R3C protein is shown on the far-left lane (lane 1, 40 kDa). Two marker lanes are in the middle with their molecular weight (kDa) indicated. Figure 6. This drawing is to provide the actual color and fluorescence properties of the purified R3 protein. The UV absorbance or the excitation for fluorescence is shown as dotted line. The fluorescence emission is shown as solid line. This R3 protein has intense red color (maximum UV absorbance at 555 nm), and strong fluorescence (excitation maximum at 555 nm and emission maximum at 590 nm). Figure 7. This drawing is to disclose the protein sequence of the R3C protein. In our example, we have the R3 protein at N-terminus, followed by the fusion part (nanobody C), with a his-tag at the C-terminus. The R3 protein and the fusion part is joined with a short linker. A skilled person can change the linker, or the order between the R3 protein core sequence and the fusion part; and add additional sequences or tags for its expression.

Detailed description

The current invention describes the generation and characterization of cysteine-free red fluorescent proteins and their uses in fusion with another protein. Lock of cysteines in the fluorescent protein renders it much more stable due to resistance to oxidation and to oligomer-formation. The intense red color makes it very visible on light background and suitable for visualization with naked eyes. Its fusion with antibodies or other bioactive proteins will find many uses in scientific research and disease diagnostics. The fluorescent protein mRuby3 is a monomeric fluorescent protein reported with relative high extinction coefficient at 128,000 at 528 nm (Bajar, B. T., E. S. Wang, A. J. Lam, B. B. Kim, C. L. Jacobs, E. S. Howe, M. W. Davidson, M. Z. Lin and J. Chu (2016). "Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting." Sci Rep 6: 20889.). It has origin from Entacmaea quadricolor, and no study has been done to replace its two cysteines at C30 and C176. Several X-ray structures of fluorescent proteins have been solved, and they are single domain protein and have a common beta barrel structure with the chromophore sitting inside the barrel. Both cysteines of mRuby3 are in the middle of beta-sheets, facing inward, according to our modeling based on the X-ray structure of mCherry 2h5q. Although most fluorescent proteins have 1-3 cysteines, the cysteine residues are chemically active. The cysteines can be oxidized to impact the structural integrity or react with another cysteine intramolecularly or intermolecularly to form unintended disulfide bonds to complicate analysis and purification. For example, cysteine-containing proteins usually form complex mixtures of proteins in different sizes when expressed in E. coli or during protein in vitro refolding. Oligomer-formation inside cells also complicates analysis (Costantini, L. M., M. Baloban, M. L.

Markwardt, M. Rizzo, F. Guo, V. V. Verkhusha and E. L. Snapp (2015). "A palette of fluorescent proteins optimized for diverse cellular environments." Nat Commun 6: 7670.). To overcome these problems, cysteine-free fluorescent proteins have been generated and showed improvements (Costantini, L. M., M. Baloban, M. L. Markwardt, M. Rizzo, F. Guo, V. V. Verkhusha and E. L. Snapp (2015). "A palette of fluorescent proteins optimized for diverse cellular environments." Nat Commun 6: 7670.). However, no cysteine-free red fluorescent protein mRuby3 or other red fluorescent proteins had been produced and tested. The impact of removal of their two naturally existing cysteines in mRuby3 on its stability and color and fluorescence properties was unknown. Cysteine replacement in other oxidation resistant fluorescent proteins showed that Cys can be replaced with Ser, Ala, and Val. There is no rule for which is better than the other, and each needs to be tested. According to Nagano et al. (Nagano, N., M. Ota and K. Nishikawa (1999). "Strong hydrophobic nature of cysteine residues in proteins." FEBS Lett 458(1): 69-71.), although similar in size, Ser may not be always a good replacement for Cys due to its hydrophilic nature. Based on our own structural analysis, what we decided to do was to replace the two cysteines with two valines, to produce the new cysteine-free fluorescent protein cf-mRuby3 (C30V, C176V) (we call it R3 protein). We constructed a highly efficient E. coli expression system with a his tag at its C-terminus. The R3 protein expresses at high level and easily purified on nickel column. It is highly soluble and has intense red color with absorbance maximum at 555 nm and fluorescence emission at 590 nm. The R3 protein (239 AA, 27 kDa) is small enough to be easily fused to another protein with its intense red color as a visible tracer. To demonstrate the feasibility, we fused the R3 gene with that of a nanobody that has affinity towards insecticide carbaryl (Liu, Z., K. Wang, S. Wu, Z. Wang, G. Ding, X. Hao, Q. X. Li, J. Li, S. J. Gee, B. D. Hammock and T. Xu (2019). "Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy-chain antibody domain." J Sci Food Agric 99(9): 4383-4390.), to create the fusion protein R3C (364 AA, 40 kDa), as an example. The R3C protein was colored, highly expressed, and easily purified to homogeneity. Omission of the easily oxidizable cysteines in R3 protein, its intense red color and fluorescence, high solubility, and good stability, make it an ideal tracer in many biological applications.

The main advantages of the present invention include

(a) The fluorescent protein is oxidation-resistant, (b) The fluorescent protein is free from oligomer-formation, (c) This fluorescent protein has an intense red color that is easily visible on light background, (d) The protein is fluorescent with excitation maximum at 555 nm and emission maximum at 590 nm.

The present invention is further described in combination with specific embodiments. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1: Location of cysteines in mRuby3 for mutation by sequence alignment and structural analysis. mCherry is a fluorescent protein with high sequence homology with mRuby3 (Figure 1), and its X-ray structure has been reported with the PDB access code of 2h5q. mRuby3 has two cysteines, C30 and C176. Many residues before and after these two cysteines are identical between mCherry and mRuby3, so that it is reasonable to assume that C30 in mRuby3 occupies the position of 144 in mCherry and C176 occupies the position of A180 in mCherry. Therefore, both cysteines (C30 and C176) of mRuby3 project towards inside as shown in Figure 2. There are limited spaces to replace both cysteines with two valines with similar hydrophobic properties, resulting our newly designed red fluorescent protein R3 (Figures 2 and 3).

Example 2: Expression and purification of the red fluorescent protein R3. We replaced two cysteines (C30, C176) in mRuby3 with two valines, to create the R3 protein, and the protein sequence is shown in Figure 3. We decided to express the R3 protein in E. coli, so that we designed a synthetic gene with codons optimized for E. coli expression. Additionally, to facilitate its purification, a his-tag was added to its C-terminus. This protein was highly expressed under a T7 promoter in E. coli strain BL21(DE3) and easily purified on nickel affinity column. One desirable feature of this protein is its intense red color, seen even before breaking the cells and clearly seen when purified the protein from mixtures on the column (Figure 4). The movement of the red protein during purification on column was easily visible. The protein is highly soluble with its correct molecular weight (27 kDa), as assessed by comparing with markers on PAGE gel (Figure 5). It has the excitation maximum at 555 nm and emission maximum at 590 nm (Figure 6).

Example 3: Expression and purification of the fusion proteinR3C. The nanobody C is a nanobody reported in the literature with affinity for insecticide Carbaryl. This nanobody was obtained from immunized alpaca and has been used to establish an immunoassay for the detection of carbaryl in cereal for food safety (Liu, Z., K. Wang, S. Wu, Z. Wang, G. Ding, X. Hao, Q. X. Li, J. Li, S. J. Gee, B. D. Hammock and T. Xu (2019). "Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy-chain antibody domain." J Sci Food Agric 99(9): 4383-4390.). The R3 protein has the intense red color and its fusion with nanobody C will colorize the nanobody and demonstrate the feasibility of coloring a bioactive protein by making the fusion protein. Of cause, beside color detection, fluorescence of the R3 protein can be used for tracing in a similar situation. The fusion was constructed by placing the R3 protein at the N-terminus, and the nanobody C as the fusion part at the C-terminus, with a short linker of GGG in between. The protein sequence of the fusion protein R3C is shown in Figure 7. Similar to the purification of the R3 protein, the R3C protein was purified with the colored protein visible during purification process. The purified protein has the expected molecular weight (40 kDa) when analyzed on PAGE (Figure 5).

Although the present invention has been described in considerable details with reference to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained therein.

SEQUENCE LISTING 07 Oct 2020

<110> Next Medicine Co., Ltd.

<120> Oxidation resistant red fluorescent proteins and use thereof

<130> temp 2020102633

<160> 2

<170> PatentIn version 3.5

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Claims (6)

Editorial Note 2020102633 There is only one page of the claim Claims

1. A red fluorescent protein, wherein said protein has its cysteine or cysteines replaced by other natural amino acids. The absence of cysteines in the protein makes it oxidation resistant and oligomer-formation resistant.

2. A red fluorescent protein, wherein said protein contains a protein sequence of SEQ ID NO: 1 and its cysteines C30 and C176 replaced individually or in combination by the preferred residues Ser, Ala, Val, and Met.

3. A red fluorescent protein, wherein said protein contains the sequence of SEQ ID NO: 2, or said protein has sequence identity of 90% or more to SEQ ID NO: 2.

4. A fusion protein, wherein said fusion protein contains: (a) the red florescent protein according to claim 1, claim 2, or claim 3; and (b) a fusion part comprising antibody, enzyme, transporter, structural protein, and transcription factor.

5. A method for producing the red fluorescent protein according to claim 1, claim 2, or claim 3; or the fusion protein according to claim 4; comprising steps of: (a) constructing their expression system under a condition suitable for producing said proteins, thereby obtaining a culture containing said proteins; and (b) isolating or recovering said proteins from said culture.

6. Use of the red fluorescent protein according to claims, claim 2, or claim 3; or the fusion protein according to claim 4; as a tracer for location and quantitation.

Figures 2020102633

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