Patent Grant
10479815
Not Applicable
Not Applicable
A sequence listing having the file name “Arjmand_SL_ST25.txt” created by Patent-in 3.5.1; on Jul. 27, 2018; and having a size of 16 kilobytes is filed in computer readable form concurrently with the specification. The sequence listing is part of the specification and herein incorporated by reference in its entirely.
The present invention relates to a method for purification and/or recovery of recombinant proteins expressed in recombinant cells. The recombinant proteins include any pharmaceutical and industrial useful proteins and are expressed as fusion with a hyrophobin-intein tag. In particular, the invention relates to a method for recovery of the intact recombinant protein from the hydrophobin-intein tag and other medium components using alcohol precipitation.
Advances in different protein expression systems ensure the recombinant production of almost every protein of interest. However, these products, especially those with pharmaceutical applications, require multiple costly subsequent purification steps and each step can inevitably decrease the final yield of the product. A protein purification process could accounts for up to 80% of production cost in the manufacturing of recombinant protein which require significant downstream processing (Walsh, 2014). Therefore, optimization of recombinant protein purification processes for reliable, simple, and cost-effective strategies with the least number of steps is one of the main issues in the industrial biotechnology and bio-separation technology development.
Fusion of affinity tags to the target protein is a commonly used method to aid protein purification as well as improving protein expression, stability, and solubility. A wide range of fusion tags with different sizes and characteristics are available; for example, E. coli maltose-binding protein tag (MBP), the Schistosoma glutathione S-transferase tag (GST), and the polyhistidine tag (Terpe, 2003). The presence of affinity tags may interfere with the structure and/or activity of the target protein; or in some cases, like for clinical applications, is unwanted. Therefore, they are usually desired to be removed from the final product in a way to produce native proteins without any extraneous sequences. At present, different approaches are available for tag removal that involves using expensive proteases or hazardous chemicals. These processes are usually non-specific and inefficient, imposing further steps of chromatography for protein purification, which increase the cost and decrease the yield of protein products.
Based on a recently discovered self-splicing protein element known as intein, the need for proteolytic processing of the fusion protein can be avoided and the tag would be removed from the target protein in a controlled manner (Wood and Camarero, 2014).
An intein is a naturally occurring segment of a protein that is involved in protein splicing. Protein splicing is a post-translational process in which an internal segment of the protein, named intein, catalyzes its excision from a protein precursor and ligation of flanking regions, named exteins, resulting in production of two proteins (Wood and Camarero, 2014; Guan et al., 2013). Sequence alignment of inteins reveals that most inteins contain a cysteine (Cys), Threonine (Thr), or serine (Ser) residue at the N-terminus; and an asparagine (Asn) or glutamin (Gln) at the C-terminus, with histidine (His) as the penultimate residue. These residues are necessary in the splicing mechanism and act as nucleophiles to create an N—S or N—O acyl rearrangement depending on the residue. This forms a linear thioester or ester intermediate. It has been reported that the cleavage of Ssp DnaB mini-intein is affected by the amino acid directly adjacent to the C-terminal of the intein. Usually Cysteine (Cys), Serine (Ser), or Threonine (Thr) directly follow the intein C-terminus as the first residue of the C-extein (+1), with Cys being the most prevalent. The side chains of these residues attack the ester bond formed in the previous step resulting in transesterification. Intein excised following the peptide bond cleavage, coupled to succinimide formation at the conserved Asn/Gln residue in downstream splice junction. Spontaneous O—N acyl rearrangement leads to formation of peptide bond between two exteins. Wood et al. (2000) indicated that presence of Cys/Thr/Ser residues at the +1 position is not necessary for C-terminal cleavage, but their occurrence is able to modulate the cleavage rate. Therefore, for C-terminal cleavage, mutation of the first residue of the C-extein is not constrained.
Structural analysis suggests that inteins are generally composed of an endonuclease protein domain and a self-splicing mini-intein domain. The endonuclease domain is not necessary for splicing. Indeed, the endonuclease domain can be deleted to form a functional splicing mini-intein. For example, by deletion of the entire endonuclease domain from the Mycobacterium tuberculosis recA intein gene, this intein was reduced form a 440 amino acid protein to a functional mini-intein with 168 amino acids (Shemella et al., 2007; Shah and Muir, 2014).
Self-cleaving affinity tags are a special group of fusion tags that possess inducible proteolytic activity combined with an appropriate affinity. They enable target protein purification and separation to be achieved in a few steps, saving time and cost. The development of pH and temperature-controllable splicing inteins has further improved the simplicity and economic advantages of this technology. One example for pH and temperature-inducible splicing inteins is Ssp DnaB mini-intein. The DnaB gene, encoding for DNA helicase of the cyanobactrium synechocystis sp. Stain PCC 6830 (Ssp), contains an intein of 429 amino acid residues. Deletion of the central 275 amino acid residues resulted in a splicing-proficient of minimal intein (Ssp DnaB mini-intein) consisting of the N-terminal 106 residues and the C-terminal 48 residues (Topilina and Mills, 2014).
Intein function can be modified from splicing to cleavage by replacing the conserved residue at C or N-terminal. For example, replacing N-terminal Cys with Ala abolishes the N-terminal cleavage and eliminates the splicing. In this case the C-terminal cleavage is observed. On the other hand, replacing the Asn in the C-terminal with Ala results in N-terminal cleavage. Blocking the splicing steps allowed the development of self-cleaving affinity tags (Shah and Muir, 2014).
The use of intein-mediated purification of proteins have been disclosed in different patent applications. Intein-mediated protein purification, using in vivo expression of an aggregator protein, was reported in patent application publication U.S. Pat. Appl. Pub. No. US 20060141570 A1; wherein the aggregator protein could be one or more phasins that was capable of specific association with granules of polyhydroxyalkanoate (PHA). The aggregator protein formed a complex with an insoluble PHA granule with low solubility using a solvent (non-alcohol) and separated from the cell lysate by centrifugation. The purification of the mentioned invention was followed by an additional downstream purification step for separation of intein-aggregator tag from the target protein in which cleavage could be controlled by pH, temperature, salt concentration, or free sulfhydryl concentration.
Tag removal remains an expensive and challenging issue in protein purification when an intact protein is needed. Inteins are used for controllable tag removal by inducing auto-cleavage as a separate step at the end of purification process. Cleavage is induced by providing preferred cleavage conditions, such as reducing agent, pH, and temperature, wherein the tag is separated from the target protein.
Du et al. (2007) demonstrated that by using an intein cleavable polyhydroxyalkanoate (PHA) synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin. The polyhydroxyalkanoate inclusions were then separated from contaminating host proteins via simple PHA bead isolation steps. Finally the host proteins were purified by specific release into the soluble fraction induced by a pH reduction.
Chen et al., in patent application publication U.S. Pat. Appl. Pub. No. 20150353597 A1, used a method for purifying a protein of interest fused to the C-terminus of an intein C-fragment with a fusion protein comprising an intein N-fragment and a purification tag. The fusion protein first purified from other proteins, and the cleavage of protein of interest from intein C-fragment was then induced to release the protein of interest. In this case, separation of purification tag and intein C-fragment form the protein of interest was done in another step of purification.
Previous intein-mediated purification methods disclosed in different patent applications, can still benefit from reducing the number of purification steps and expensive protein separation using affinity columns.
Hydrophobins are a group of small surface-active proteins produced by filamentous fungi. Hydrophobins are relatively small (approximately 100 amino acids), contain eight disulfide-forming Cys residues in a conserved pattern, and could self-assemble on interfaces. Using hydrophobin as self-assembly tags are applicable and has many advantages such as considerable selectivity, high yield, and being simple to perform hydrophobin aggregation technically (Linder et al., 2004).
Hydrophobins which are identified to date are generally classified as either class I or class II. Both types have been identified in fungi as secreted proteins that self-assemble at interfaces into amphipathic films. Assemblages of class I hydrophobins are generally relatively insoluble whereas those of class II hydrophobins readily dissolve in a variety of solvents. The hydrophobins of interest to us are the class II from Trichoderma reesei. The hydrophobins number I and II (HFBI and HFBII) of Trichoderma reesei have been most extensively studied. Both have a size of about 7.5 KDa and are widely used in molecular studies (Joensuu et al., 2010; Linder et al., 2001).
Alcohol precipitation has been used in different purification methods including purification of hydrophobin as the target protein as described in patent application publication No, 20150057434 A1. However, alcohol precipitation has not been used for purification of other proteins when the hydrophobin is used as the fusion tag in combination with intein (intein-hydrophobin tag) to purify an intact protein.
It is the object of the present invention to provide a protein purification system which can operate rapidly, efficiently, and cheaply; for example, avoiding using expensive materials such as affinity columns and proteases as well as reducing the number of purification steps; thereby, overcoming the disadvantages of currently employed protein purification systems, such as involving too many steps, complicated manipulation, and high expenses.
The invention is directed to a method of purifying a target protein from a cell culture supernatant. The cell culture supernatant includes a recombinant fusion protein comprising a target protein domain, a self-cleaving intein, and a hydrophobin protein domain. The intein is located between the target protein domain and the hydrophobin protein domain. The hydrophobin protein domain is optionally linked by an amino acid linker to a self-cleaving intein. The method of producing the cell culture supernatant comprises nucleic acids encoding the recombinant fusion protein, constructing plasmids carrying the nucleic acids, cells stably transfected with the plasmids, and culturing the cells.
In a preferred embodiment, the invention is directed to a method of purifying a target protein from a cell culture supernatant comprising:
The system and method of the invention provide many advantages and new features including the following:
(1) Hydrophobin as the purification tag is used in combination with intein for purification of intact proteins.
(2) Alcohol precipitation is used for protein purification using hydrophobin as a fusion tag.
(3) There is no need for an extra step of fusion tag cleavage and separation, and it happens spontaneously after centrifuging the second stirred solution in step (h) above, therefore, reducing the number of purification steps.
A fuller understanding of the nature and objects of the present invention will become apparent upon consideration of the following detailed description taken in connection with the accompanying drawings, wherein:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and as described in the literature, including books in cell biology, chemistry, and molecular biology (For example: Walsh, 2014; Terpe, 2003; and Wood and Camarero, 2014).
As used herein, the term C1-C3 alcohol refers to an alcohol selected from a group consisting of C1, C2, and C3 alcohols, preferably Methanol (C1), Ethanol (C2), and Propanol (C3). The volume fraction of an alcohol added to a solution, expressed as percentage, is the ratio of the volume of the alcohol divided by the sum of the volumes of the alcohol plus the volume of the solution the alcohol to be added measured separately. For example, to make a 50% v/v ethanol solution, you would measure 50 ml of ethanol and separately measure 50 ml of a solution, then mix the two together.
The terms recombinant fusion protein and fusion protein are used interchangeably and may also be sometimes referred to as hydrophobin-intein-target protein, preferably HFBII-intein-target protein. The terms recombinant target protein and target protein are used interchangeably. The term “cell culture supernatant” is used to address the solution containing the fusion protein at the start of the purification process and other supernatants obtained during the purification process are addressed by other terms such as first supernatants, second supernatants, etc.
In this invention we describe (1) a preparation process of a cell culture supernatant containing a recombinant fusion protein; and (2) a purification process of the recombinant target protein from the cell culture supernatant containing said recombinant fusion protein. These processes are illustrated in examples 1 to 5 in the section of examples.
The recombinant fusion protein comprises a target protein domain, a self-cleaving intein, and at least one hydrophobin protein domain. The intein is located between the target protein domain and the hydrophobin domain. Optionally, the hydrophobin domain could covalently be attached to the intein by an amino acid linker. The hydrophobin is hydrophbin I (HFBI) or hydrophobin II (HFBII), preferably hydrophobin II from Trichoderma reesei. The intein is preferably Ssp DnaB mini-intein from Synechocystis Sp. Pcc 6803. The target protein can be any recombinant protein from animals, plants, yeasts, or bacteria, for example, human vascular endothelial growth factor A 165 (VEGFA165) or human alpha 1-antitrypsin (A1AT).
(1) Preparation of the cell culture supernatant: The cell culture supernatant is prepared by introducing an expression vector comprising a nucleic acid encoding said fusion protein into a host cell followed by culturing said host cell, illustrated in examples 1 to 5. The host cell may be derived from prokaryotes or eukaryotes, preferably a yeast cell of a strain from Pichia pastoris.
The vector is prepared by constructing an expression plasmid, preferably plasmid pPICZA (Example 1); amplifying it in a cell, preferably in E. coli (Example 2). The host cell, preferably Pichia pastoris, transfected with the plasmid is cultured forming a recombinant cell culture medium (Example 3), wherein the fusion protein is expressed in a plurality of the host cells (Example 4). The fusion protein is allowed to leave the host cells either by cell secretion or cell lysis into the recombinant cell culture medium. The cell culture supernatant is formed preferably by centrifuging the recombinant cell culture medium, and it is then separated, preferably by decanting. The cell culture supernatant containing the fusion protein is then used in the purification process of the target protein.
(2) Purification process:
Part I comprises adding a C1-C3 alcohol, preferably from about 50% (v/v) to about 75% (v/v), more preferably 66.66% (v/v) to 71.5% (v/v) of C2 alcohol, to the cell culture supernatant, making a first alcohol solution; stirring the first alcohol solution, preferably for 15-45 min and preferably at room temperature, forming a first stirred solution; centrifuging the first stirred solution, preferably for 30-45 min at 1300-1500 g, forming a first precipitate containing the cell residue and a first supernatant solution comprising a substantial amount of the fusion protein. The first supernatant solution is decanted and separated.
Part II comprises adding a C1-C3 alcohol, preferably about 50% (v/v) to about 66.66% (v/v) of C2 alcohol, and more preferably 54.54% (v/v) of C2 alcohol, to the first supernatant solution from Part I making the second alcohol solution; stirring the second alcohol solution, preferably for 15-45 min, preferably at room temperature, forming a second stirred solution; centrifuging the second stirred solution, preferably for 30-45 min at 1300-1500 g, wherein the target protein separates from the fusion protein and precipitates, forming a second precipitate containing the target protein and a second supernatant. The second supernatant is decanted and the second precipitate containing the target protein is then separated to yield a substantial amount of the purified target protein.
One of the advantages of the purification procedure described in this invention is that there is no need for an extra step of fusion tag cleavage and separation, while this happens spontaneously in the second alcohol purification step described in Part II.
The present invention will be illustrated in more details with reference to the following examples. Examples 1 to 5 describes an embodiment related expression and purification of VEGFA165 as a target protein. The processes described in examples 1 to 5 are then repeated in another embodiment related to expression and purification of A1AT as a target protein. These examples are presented only for illustrative purpose and are not intended to limit the scope of the present invention in any way.
VEGFA165 from human (SEQ ID No: 4) as a target protein
For constructing hydrophobin-intein-VEGFA165; the sequences of protein HFBII from Trichoderma reesei with its native signal peptide (SEQ ID No: 1); Ssp Dnab mini-intein from Synechocystis Sp. Pcc 6803 with an optional additional Cystein amino acid at the C-terminal for improving splicing (SEQ ID No: 2); an optional linker with 12 amino acids at the N-terminal (SEQ ID No: 3); and VEGFA165 from human (SEQ ID No: 4) were converted to the nucleotide sequence according to the codon bias of yeast Pichia pastoris. The sequence with restriction site for EcoRI/KpnI restriction enzyme at the 5′ and 3′ end and kozak sequence of AOX1 gene from Pichia pastoris, after EcoRI restriction recognition site and just before beginning ATG, was chemically synthesized (SEQ ID No: 5).
The synthetic sequence was cloned between EcoRI and KpnI restriction site behind the AOX1 promoter in the pPICZA plasmid (
pPICZA vector carrying expression construct for HFBII-intein-VEGFA165 chimeric gene was transformed to E. coli (strain DH5α) and recombinant clones were selected. The transformants were plated on low salt LB agar plates containing Zeocin® (25 μg/ml) for selection. The positive clones were inoculated in 5 ml low salt medium supplemented with Zeocin® (25 μg/ml) and incubated over night at 37° C. The amplified recombinant plasmid pPICZA containing DNA sequence of HFBII-intein-VEGFA165 construct was extracted from the bacteria and sent for sequencing to confirm the proper sequences.
Recombinant vector pPICZA, containing DNA sequence of HFBII-intein-VEGFA165 construct, was linearized with SacI in the AOX1 promoter region to stimulate homologous recombination when the plasmid is transformed into the Pichia pastoris competent cells. Linearized DNA were integrated into AOX1 promoter region of Pichia pastoris to give the desired strain. To make competent cells, Pichia pastoris (X33) was cultured overnight in 50 mL of YPD medium at 30° C. 500 mL of YPD medium was inoculated with 0.1-0.5 mL of the cultured medium in a 2 L flask and incubated at 30° C. to reach an OD600 of 1.3-1.5. After centrifugation at 1500 g at 4° C. for 5 min, the pellet was dissolved in 500 mL of cold sterilized deionized water. After re-centrifugation under the same conditions, the pellet was dissolved in 250 mL of cold sterilized water. The same procedure was repeated with 20 mL and then 1 mL of cold 1 M sorbitol to give competent cells at a final volume of 1 mL. 80 μL of the competent cell was mixed with 1-5 μg of recombinant vector pPICZA linearized with SacI and incubated in an electroporation cuvette for 5 min on ice. The competent cells electroporated at 1500 volt, 200Ω and 25 UF and immediately were re-suspended in 1 ml of 1 M sorbitol and transferred to sterilized microcentrifuge tubes. The transformant were plated in YPD agar supplemented with Zeocin® (100 μl/ml) and grown for 3 days at 30° C.
The genomic DNAs of transformant were extracted and analyzed for the correct integration of HFBII-intein-VEGFA165 construct in yeast chromosome using PCR with the 5′ AOX1 primer (5′-GACTGGTTCCAATTGACAAGC-3′) and 3′ AOX1 primer (5′-GCAAATGGCATTCTGACATCC-3) and sequencing of PCR results.
DNAs amplified by the PCR were found to be about 1.5 Kb in size, as indicated by agarose gel electerophoresis (
Colonies of transformant Pichia pastoris carrying HFBII-intein-VEGFA165 sequence were used for inoculation of 25 ml of YPG in a 250 ml flask and grown at 28-30° C. in a shaking incubator (250-300 rpm) until culture reached an OD600 of 2-6 (approximately 16-18 hours). The cells were harvested by centrifuging at 1500 g for 5 min and re-suspended in YPM (containing 0.5% methanol to induce the expression and pH was set at 7.8) to reach an OD600 of 1. Methanol was added to the culture every 24 hours for 96 hours to maintain induction (1% at the first day and 0.5% in the next days of induction). The culture was then centrifuged at 3000 g for 5-10 min, and the supernatant was transferred to a separate tube for analysis of protein expression by silver nitrate stained SDS-PAGE and ELISA.
ELISA was conducted to measure the quantities of the expressed chimeric protein HFBII-intein-VEGFA165 using human VEGF DueSet ELISA kit (R & D systems, DY293B-05) according to the manufacturer's protocol. The ELISA experiment detected the recombinant secretory VEGFA165 with concentration of 53 mg/mL in the culture supernatant.
To observe the secretory expressed protein, 10 μL of supernatant were subjected to 12% SDS-PAGE. The proteins were visualized as bands by staining with silver nitrate to determine whether the recombinant chimeric protein were expressed and secreted (
To purify recombinant VEGFA165 (fused with HFBII using intein) from the supernatant of the yeast culture medium (cell culture supernatant), 50% (v/v) to 75% (v/v) of C1-C3 alcohol was added to the cell culture supernatant (first alcohol solution), while 66.66% (v/v) of C2 alcohol was preferable. The solution was stirred at room temperature for 30 min (first stirred solution). The mixture (first stirred solution) was centrifuged for 30-45 min at 14000 g (first centrifugation) and the supernatant containing the fusion protein (first supernatant) was decanted into a clean vials. From 50% (v/v) to 66.66% (v/v) of C1-C3 alcohol was added to the first supernatant (second alcohol solution), while 54.54% (v/v) of C2 alcohol preferable. The solution was stirred for 30 minutes at room temperature (second stirred solution) and centrifuged for 30-45 minutes at 14000 g (second centrifugation). The condition of experiment in second stirring and centrifugation appeared to induce the autolytic property of used intein and the VEGFA165 was separated from the rest of construct and precipitated. The hydrophobin and fused intein stayed in the alcohol solution and separated from the precipitate. The obtained precipitate consisted of the target protein (VEGFA165) was dissolved in water and subjected to the SDS-PAGE to be visualized. The results of SDS-PAGE indicated that the VEGFA165 was separated from the other components with a high purity (
Human A1AT (SEQ ID No: 6) as a target protein
The expression and purification processes for A1AT protein were performed by repeating the examples 1 to 5 while substituting VEGFA165 protein with A1AT protein, including:
The results of ELISA experiments verified the presence of recombinant A1AT in the dissolved solution of the second precipitate and its absence in the solution of the first precipitate.
The activity of the produced recombinant A1AT in the cell culture supernatant and the first and second precipitations was measured by elastase inhibitory capacity (EIC) according to Tavasoli et al. (2017). The results of EIC indicated that recombinant A1AT was in active form in supernatant of cell culture. The presence of elastase inhibitory activity was detected in the second precipitation while no elastase inhibitory activity was seen in the first precipitation.
While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention as claimed.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20060141570 | Wood | Jun 2006 | A1 |
| 20150057434 | Schelle | Feb 2015 | A1 |
| 20150353597 | Chen | Oct 2015 | A1 |
| Entry |
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| Kirkland et al. (J Ind Microbiol Biotechnol (2011) 38:327-335). |
| Mills et al. (The Journal of Biological Chemistry vol. 289, No. 21, pp. 14498-14505, May 23, 2014). |
| Du J and Rehm BHA, “Purification of target proteins from intracellular inclusions mediated by intein cleavable . . . ”, Microb Cell Fact, 16:2553-2560, (2017). |
| Guan D, et al., “Split intein mediated ultra-rapid purification of tagless protein (SIRP)”, Biotechnol Bioeng, 110:2471-2481, (2013). |
| Linder MB, et al., “Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems”, Biochem, 43:11873-11882, (2004). |
| Number | Date | Country | |
|---|---|---|---|
| 20180371011 A1 | Dec 2018 | US |
