Protein purification using intein-hydrophobin tag and alcohol precipitation

Abstract

A method of downstream purification of a recombinant protein in fusion with a hydrophobin-intein tag using alcohol precipitation. The method comprises (1) constructing a plasmid expressing a fusion protein in a host cell, wherein the fusion protein includes a target protein domain, an intein, and a hydrophobin domain; (2) culturing the host cell transfected with the plasmid forming a cell culture medium; (3) separating a cell culture supernatant containing the fusion protein from the cell culture medium; and (4) purifying a substantial amount of the target protein from the cell culture supernatant without its hydrophobin-intein fusion tag using alcohol precipitation.

Claims

1. A method of purifying a target protein, the method comprising: (a) preparing a cell culture supernatant comprising a fusion protein, wherein the fusion protein comprising: (i) a target protein domain; (ii) a self-cleaving intein; (iii) at least one hydrophobin protein domain, wherein the intein is located between the target protein domain and the hydrophobin domain; (b) adding a C1-C3 alcohol to the cell culture supernatant making a first alcohol solution; (c) stirring the first alcohol solution forming a first stirred solution; (d) centrifuging the first stirred solution forming a first precipitate and a first supernatant, wherein the first supernatant comprising the fusion protein; (e) separating the first supernatant comprising the fusion protein; (f) adding a C1-C3 alcohol to the first supernatant making the second alcohol solution; (g) stirring the second alcohol solution forming a second stirred solution; (h) centrifuging the second stirred solution forming a second supernatant and a second precipitate, wherein the second precipitate comprising the target protein; and (i) separating the second precipitate comprising the target protein to yield a substantial amount of the purified target protein, wherein the intein is Ssp DnaB mini-intein from Synechocystis, wherein the volume fraction of C1-C3 alcohol added in making the first alcohol solution in step (b) is from about 50% (v/v) to about 75% (v/v), and wherein the volume fraction of the C1-C3 alcohol added in making the second alcohol solution in step (f) is from about 50% (v/v) to about 66.66% (v/v).

2. The method of claim 1, wherein the cell culture supernatant is prepared comprising the steps: (i) constructing a plasmid expressing the fusion protein in a host cell; (ii) culturing the host cell transfected with the plasmid forming a recombinant cell culture medium, wherein the fusion protein is expressed in a plurality of the host cells; (iii) allowing the fusion protein to leave the host cells either by cell secretion or cell lysis into the recombinant cell culture medium; (iv) centrifuging the recombinant cell culture medium forming the cell culture supernatant comprising the fusion protein; and (v) separating the cell culture supernatant to be used for the purification of the target protein.

3. The method of claim 1, wherein the intein undergoes c-terminal cleavage as a result of a shift in pH and temperature.

4. The fusion protein of claim 1, wherein the intein comprises an additional Cystein amino acid at its C-terminal for improving splicing.

5. The fusion protein of claim 1, wherein the at least one hydrophobin domain is covalently attached to the intein by an amino acid linker.

6. The method of claim 1, wherein the target protein is vascular endothelial growth factor A.sub.165.

7. The method of claim 1, wherein the target protein is human alpha 1-antitrypsin.

8. The method of claim 1, wherein the hydrophobin is hydrophobin number II from Trichoderma reesei.

9. The method of claim 1, wherein the first supernatant is separated from the first precipitate by decanting the first supernatant.

10. The method of claim 1, wherein the second precipitate is separated from the second supernatant by decanting the second supernatant.

11. The method of claim 1, wherein the volume fraction of C1-C3 alcohol added in making the first alcohol solution in step (b) is about 66.66% (v/v), and the alcohol is C2 alcohol.

12. The method of claim 1, wherein the volume fraction of the C1-C3 alcohol in making the second alcohol solution in step (f) is about 54.54 (v/v), and the alcohol is C2 alcohol.

13. The method of claim 2, wherein the host cell is a strain from Pichia pastoris.

14. The method of claim 2, wherein the cell culture supernatant is separated by decanting the cell culture supernatant.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) 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:

(2) FIG. 1 shows a flow chart of a general protocol for purification of a target protein;

(3) FIG. 2 is a schematic shape showing recombinant plasmid pPICZA containing HFBII-intein-Gene Of Interest (GOI) sequence between EcoRI/KpnI restriction sites;

(4) FIG. 3 is an agarose gel electrophoresis showing PCR amplification of (a) HFBII-intein-VEGFA.sub.165 and (b) HFBII-intein-A1AT constructs with AOX1 primers. The related 1.5 kb and 2 kb PCR bands were observed in agarose gel electrophoresis;

(5) FIG. 4 is a SDS-PAGE showing the expression of recombinant (a) HFBII-intein-VEGFA.sub.165 and (b) HFBII-intein-A1AT. Lane 1a) control negative containing the supernatant of non-recombinant Pichia pastoris. Lane 2a) protein marker. Lane 3a) the supernatant of recombinant Pichia pastoris expressing HFBII-intein-VEGFA.sub.165. Lane 1b) protein marker. Lane 2b) the supernatant of recombinant Pichia pastoris expressing HFBII-intein-A1AT. The cleaved HFBII-intein is observed in 33 kD. The recombinant VEGFA.sub.165 and A1AT is observed in 23 and 50 kD respectively. The intein cleavage was triggered during the SDS-PAGE preparation and loading;

(6) FIG. 5 Purification of VEGFA.sub.165 and western blot of purified protein. Lane 1a) the first precipitation of purification, containing all protein of media except target protein. Lane 2a) the second precipitation of purification, containing the purified target protein (VEGFA.sub.165). Lane 3a) supernatant of recombinant yeast cell culture expressing VEGFA.sub.165 without HFBII-intein fusion partner as the positive control. Lane 1b) western blot of the first precipitation of purification, containing all protein of media except target protein. Lane 2b) western blot of the second precipitation of purification, containing the purified target protein (VEGFA.sub.165). Lane 3b) supernatant of recombinant yeast cell culture expressing VEGFA.sub.165 without HFBII-intein fusion partner as the positive control;

(7) FIG. 6 is a SDS-PAGE showing purified A1AT. Lane 1) supernatant of transformant yeast cell culture before purification. Lane 2) the second precipitation of purification containing the purified target protein (A1AT). Lane 3) protein marker.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(8) 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).

(9) 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.

(10) 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.

(11) 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.

(12) 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 (VEGFA.sub.165) or human alpha 1-antitrypsin (A1AT).

(13) (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.

(14) 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.

(15) (2) Purification process: FIG. 1 shows a general scheme for purification of a target protein. The purification process comprises two parts, Part I and Part II. Each part comprises the steps of: an alcohol addition, a stirring, a centrifugation, a precipitation, and a separation as described below and illustrated in example 5.

(16) 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.

(17) 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.

(18) 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.

(19) 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 VEGFA.sub.165 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.

(20) VEGFA.sub.165 from human (SEQ ID No: 4) as a target protein

Example 1

Construction of Expression Plasmid pPICZA for Expression of the Fusion Protein Hydrophobin-Intein-VEGF A165

(21) For constructing hydrophobin-intein-VEGFA.sub.165; 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 VEGFA.sub.165 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).

(22) The synthetic sequence was cloned between EcoRI and KpnI restriction site behind the AOX1 promoter in the pPICZA plasmid (FIG. 2).

Example 2

Transformation of HFBII-Intein-VEGFA165 in E. coli (Strain DH5)

(23) pPICZA vector carrying expression construct for HFBII-intein-VEGFA.sub.165 chimeric gene was transformed to E. coli (strain DH5) and recombinant clones were selected. The transformants were plated on low salt LB agar plates containing Zeocin (25 g/ml) for selection. The positive clones were inoculated in 5 ml low salt medium supplemented with Zeocin (25 g/ml) and incubated over night at 37 C. The amplified recombinant plasmid pPICZA containing DNA sequence of HFBII-intein-VEGFA.sub.165 construct was extracted from the bacteria and sent for sequencing to confirm the proper sequences.

Example 3

Transformation of Pichia pastoris and Selection of Pichia pastoris Transformants

(24) Recombinant vector pPICZA, containing DNA sequence of HFBII-intein-VEGFA.sub.165 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 OD.sub.600 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 L of the competent cell was mixed with 1-5 g 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, 200 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 l/ml) and grown for 3 days at 30 C.

(25) The genomic DNAs of transformant were extracted and analyzed for the correct integration of HFBII-intein-VEGFA.sub.165 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.

(26) DNAs amplified by the PCR were found to be about 1.5 Kb in size, as indicated by agarose gel electerophoresis (FIG. 3a).

Example 4

Expression of Recombinant Chimeric Protein HFBII-Intein-VEGFA165

(27) Colonies of transformant Pichia pastoris carrying HFBII-intein-VEGFA.sub.165 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 OD.sub.600 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 OD.sub.600 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.

(28) ELISA was conducted to measure the quantities of the expressed chimeric protein HFBII-intein-VEGFA.sub.165 using human VEGF DueSet ELISA kit (R & D systems, DY293B-05) according to the manufacturer's protocol. The ELISA experiment detected the recombinant secretory VEGFA.sub.165 with concentration of 53 mg/mL in the culture supernatant.

(29) To observe the secretory expressed protein, 10 L 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 (FIG. 4).

Example 5

Purification of VEGFA165 as a Target Protein (FIG. 1)

(30) To purify recombinant VEGFA.sub.165 (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 VEGFA.sub.165 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 (VEGFA.sub.165) was dissolved in water and subjected to the SDS-PAGE to be visualized. The results of SDS-PAGE indicated that the VEGFA.sub.165 was separated from the other components with a high purity (FIG. 5). The results of ELISA experiments verified the presence of recombinant VEGFA.sub.165 in the dissolved solution of second precipitate and its absence in the solution of first precipitate.

(31) Human A1AT (SEQ ID No: 6) as a target protein

(32) The expression and purification processes for A1AT protein were performed by repeating the examples 1 to 5 while substituting VEGFA.sub.165 protein with A1AT protein, including: (i) Construction of expression plasmid pPICZA for expression of the fusion protein hydrophobin-intein-A1AT, similar to Example 1, but including the sequences of HFBII (SEQ ID No: 1), intein (SEQ ID No: 2), optional linker (SEQ ID No: 3), and A1AT protein (SEQ ID No: 6) which was converted to the nucleotide sequence, based on the codon preference of Pichia pastoris, and chemically synthesized for the hydrophobin-intein-linker-A1AT (SEQ ID No: 7). (ii) Transformation of HFBII-intein-A1AT in E. coli (strain DH5), similar to Example 2; (iii) Transformation of Pichia pastoris and selection of Pichia pastoris transformants, similar to Example 3. In this case, DNA amplified by the PCR was found to be about 2 Kb in size for A1AT, as indicated by agarose gel electerophoresis (FIG. 3b); (iv) Expression of recombinant chimeric protein HFBII-intein-A1AT, similar to Example 4 (FIG. 4). In this case, the measurement of expression by ELISA was performed using human serpin A1 Duoset ELISA kit (R & D systems, DY1268); and (v) Purification of A1AT protein as a target protein, similar to Example 5. The results of SDS-PAGE indicated that the A1AT was separated from the other components with a high purity (FIG. 6).

(33) 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.

(34) 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.

(35) 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.

REFERENCES

US Patent Documents

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OTHER PUBLICATIONS

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