WO2017198562A1

WO2017198562A1 - Method of producing domains of protein

Info

Publication number: WO2017198562A1
Application number: PCT/EP2017/061455
Authority: WO
Inventors: Harald Althaus; Sudipa GALGALKAR; Praveen Bhat GURPUR; Martin Hahn; Andreas Kappel; Herbert Schwarz
Original assignee: Siemens Healthcare Gmbh
Priority date: 2016-05-19
Filing date: 2017-05-12
Publication date: 2017-11-23

Abstract

The present invention relates to a method (10) for producing one or more domains of protein in their native state. The method comprises the steps of introducing one or more enzyme recognition sites (3) within a gene for a recombinant protein (2), wherein said gene for recombinant protein (2) encodes for said one or more domains of protein; forming an expression construct (4) by cloning said gene for said recombinant protein (2) into an expression vector (1); transferring said expression construct (4) into mammalian cells (5); expressing said gene for recombinant protein (2) containing one or more enzyme recognition sites (3) in said mammalian cells (5); contacting said expressed recombinant protein with an enzyme specific to said one or more enzyme recognition sites; and producing one or more domains of proteins in their native state.

Description

Title of the invention

Method of producing domains of protein Description

The present invention relates to a method of producing do^¬ mains of protein in their native state.

Recombinant proteins are widely used in biology and find their application in therapeutics, diagnostics, pharmaceuti^¬ cal industry and biological research. In some of these appli^¬ cations, expression of the entire protein is not necessary. Therefore, in such cases, only a certain domain of the pro^¬ tein is expressed. The important aspect in expressing only a certain domain of the protein is the way the expressed pro^¬ tein folds. In order that a protein function correctly, it is essential that a protein be in its native state, i.e. be folded or assembled correctly. The native state of a protein is known to be the state in which a protein is most stable. Failure to fold in its native state generally results in protein being non-functional. In certain cases, a mis-folded protein may have modified functionality or may be toxic.

The issue of mis-folding of proteins has been addressed by several methods. In one of the known methods, a different ex^¬ pression background is provided to check if the domain of the protein is folded correctly. Therefore, the required domain of the protein is expressed in a mammalian cell or an insect cell. In another known method, the recombinant protein is co- expressed with a chaperone protein that assists protein fold^¬ ing. In other known methods, varying the temperature of growth of the cells is thought to promote proper protein folding . However, the results of all the above mentioned methods are unpredictable and the possibility of producing misfolded pro^¬ tein is high. This may result in greater loss of resources and time. In light of the above, there exists a need to provide a meth^¬ od of producing domains of protein in their native state in a systematic and efficient manner.

Therefore, the object of the present invention is to provide a method of producing domains of protein in their native state that is more systematic and efficient. The invention refers to a method of producing one or more do^¬ mains of protein in their native state. One or more domains of protein in their native state comprise one or more domains of protein in a correctly folded state. According to the invention, the method comprises a step of introducing one or more enzyme recognition sites within a gene for recombinant protein. The gene of the recombinant protein encodes for one or more domains of protein. Enzyme recognition sites are protein sequences on which enzymes spe- cific to the site act. The method further comprises a step of forming an expression construct by cloning into an expression vector said gene for a recombinant protein that has one or more domains of protein. An expression vector is a

deoxyribose nucleic acid (DNA) molecule that is used for transferring foreign genetic material into a cell where such foreign genetic material may be expressed.

The method further comprises a step of transferring the ex^¬ pression construct into mammalian cells. These mammalian cells may be in a culture medium or may be present in a live animal. Mammalian cells provide an environment for expression of the recombinant protein. The mammalian cell lines that may be used include, for example, HEK 293, CHO, HeLa. The method comprises a step of expressing the gene for the recombinant protein that contains one or more genes for one or more domains of protein. On expression, the recombinant protein that has one or more domains of protein is produced in said mammalian cells.

The method further comprises a step of contacting said recom- binant protein with an enzyme specific to said one or more enzyme recognition sites. The enzyme specific to the enzyme recognition site reacts on the expressed enzyme recognition sequence, preferably cleaving the expressed enzyme recogni^¬ tion sequence, thereby separating a part of the protein from the rest of the expressed protein. Therefore, one cleaving the enzyme recognition sites, one or more domains of proteins are produced in their native state. The complete expression of the recombinant protein ensures that the recombinant pro^¬ tein is in its native state.

Additionally, the method comprises a step of purifying said one or more domains of protein from the culture medium. On proteolysis, the one or more domains of protein are cleaved from the rest of the recombinant protein. Thus said one or more domains of protein may be separated and purified for further use.

Therein, the step of introducing the one or more enzyme recognition sites within the gene for recombinant protein comprises inserting the one or more enzyme recognition sites at gene sequences encoding for one or more domains of pro^¬ tein. Therefore, on cleaving such one or more expressed en^¬ zyme recognition sites, the one or more domains of protein are separated from the rest of the expressed protein. As the one or more domains of protein are cleaved after the complete expression of the protein, the separated domains of the pro^¬ teins would be folded correctly.

Therein, the expression construct is transferred into mamma- lian cells using a transfection process. Transfection is a method of introducing genetic material into eukaryotic cells. Transfection methods that may be used for transferring the expression construct into the mammalian cells may include, for example, chemical methods, electroporation, viral trans^¬ duction, etc.

Therein, the one or more enzyme recognition sites are prote- ase recognition sites. Proteases are enzymes that perform proteolysis of protein sequences. On exposure to protease en^¬ zyme, protease recognition sites are proteolysed, thereby cleaving one or more protein domains of interest from the rest of the protein.

Therein, the enzyme is a protease enzyme. Protease enzyme recognizes the one or more expressed enzyme recognition sites in the recombinant protein and cleave such enzyme recognition sites .

Alternatively, the enzyme is a combination of protease enzyme with one or more enzymes chosen from the group of enzymes comprising hydrolases, esterases and lyases. Therein, the one or more domains of protein are purified from the culture medium using chromatographic techniques. Such chromatographic techniques may include, for example, affinity chromatography, size exclusion chromatography, etc. Therein, the gene for recombinant protein is a gene for a transmembrane protein. The transmembrane proteins, the gene for which is used, may include, but not be limited to, integrins, ion channels, transporter proteins, G protein- coupled receptors, enzyme-linked receptors, Fey receptor and its variants, and structural proteins like sarcoglycans .

Therein, the one or more enzyme recognition sites are insert^¬ ed between the gene sequences encoding for the membrane do^¬ main and the extracellular domain of the recombinant protein. Therefore, the enzyme recognition sites are at the junction between the membrane and the extracellular domains of the re^¬ combinant protein. Thus, post expression, on contacting with an enzyme specific to the enzyme recognition site, the extra- cellular domain of the recombinant protein will be cleaved from the rest of the recombinant protein. The extracellular portion of a transmembrane protein may be used in several ap^¬ plications. For example, the extracellular domains of transmembrane proteins are used as targets for drugs in phar^¬ maceutical industry. During screening of drug molecules, the binding characteristics and efficiency of the drug molecule to the protein is tested. In such circumstances, use of only the extracellular domain, i.e. the ligand binding portion of the protein is beneficial for testing the binding character^¬ istics of the drug.

The invention also refers to an expression vector for use in producing one or more domains of protein in their native state. According to the invention, the expression vector com^¬ prises one or more multiple cloning sites and one or more gene sequences encoding one or more enzyme recognition sites that are flanked on either side by said one or more multiple cloning sites. A multiple cloning site is a short segment of DNA that contains several restriction sites that can be used to insert a short piece of DNA within the multiple cloning site .

The invention also refers to a kit for producing one or more domains of protein in their native state. According to the invention, the kit comprises of one or more single type mam^¬ malian cell lines. The mammalian cell lines may be, for exam^¬ ple, CHO, HeLa, etc. According to the invention, the kit fur^¬ ther comprises of an expression vector with one or more gene sequences encoding one or more enzyme recognition sites. The expression vector may be any vector used for the purposes of cloning that is known to a person having ordinary skill in the art . According to an embodiment of the invention, the kit may fur^¬ ther comprise an expression vector with at least one gene for an enzyme specific to said enzyme recognition site. There^¬ fore, the expression vector with at least one gene for the enzyme is co-transfected with the expression vector with one or more genes for one or more enzyme recognition sites into the mammalian cell lines. According to an embodiment of the invention, the kit may fur^¬ ther comprise an enzyme specific to the one or more enzyme recognition sites.

According to another embodiment, the one or more single type mammalian cell lines constitutively express an enzyme specif^¬ ic to said one or more enzyme recognition sites. Therefore, these one or more single type mammalian cell lines continu^¬ ously produce the enzyme specific to said one or more enzyme recognition sites. This eliminates the need for introducing externally the enzyme specific to said one or more enzyme recognition sites.

The present invention is further described hereinafter with reference to illustrated embodiments shown in the accompany- ing drawings, in which:

FIG 1 illustrates a schematic diagram of an embodiment of a method according to the invention; and FIG 2 illustrates a schematic diagram of an expression vector for obtaining one or more protein domains of interest in their native states according to the invention . Hereinafter, embodiments for carrying out the present inven^¬ tion are described in detail. The various embodiments are de^¬ scribed with reference to the drawings, wherein like refer^¬ ence numerals are used to refer to like elements throughout. In the following description, for purpose of explanation, nu- merous specific details are set forth in order to provide a thorough understanding of one or more embodiments. It may be evident that such embodiments may be practiced without these specific details. FIG 1 provides an illustration of an exemplary method 10 for producing one or more domains of protein in their native state. According to the method 10, at step 11, one or more gene sequences encoding for one or more enzyme recognition site 3 are inserted within a gene for a recombinant protein 2. The recombinant protein 2 is the protein from which do^¬ mains of protein are to be obtained. In one embodiment, a gene for a protease recognition site 3 is inserted in the gene for the recombinant protein 2. In this embodiment, the gene for protease recognition site 3 is inserted at a junc^¬ tion of the gene sequence encoding the extracellular and mem^¬ brane domains of the recombinant protein 2. The gene for the recombinant protein 2 along with the gene for the protease recognition site 3 is cloned into the expression vector 1 to form an expression construct 4 in step 11.

In the next step 12 of the method 10, the expression con^¬ struct 4 is transferred into a mammalian cell line, HeLa cells 5. Alternatively, the expression construct 4 can be transfected into a live animal directly. HeLa cell type is an immortal human cell line. In the embodiment, the expression construct 4 is transfected into the HeLa cells 5. The trans- fection process is completed by viral transduction, wherein the expression construct 4 is transferred into the Hela cells 5 using a virus. The process of viral transduction is well known in the state of the art and therefore has not been de^¬ picted in FIG 1. The recombinant genes are expressed for pro^¬ tein production in the next step 13 of the method 10. The re- combinant protein produced has an extracellular domain 6 and a membrane domain 7. As the whole recombinant protein is ex^¬ pressed, the native state of the protein is achieved. There may also be post-translational modification of the recombi^¬ nant protein, for example, glycosylation, thereby ensuring normal physiological state of the recombinant protein. In the next step 14 of the method 10, HeLa cells are contacted with protease enzyme 8. The protease enzyme 8 used may be an un^¬ common protease. Alternatively, the mammalian cell line 5 may also be genetically modified to constitutively express prote^¬ ase enzyme. Such constitutively expressed protease enzyme is secreted into the culture medium. The protease enzyme 8 rec^¬ ognizes the expressed protease recognition site present in the junction between the extracellular 6 and the membrane do^¬ mains 7 of the recombinant protein. Alternatively, protease enzyme may also be used in a combination with other enzymes such as hydrolases, lyases and esterases. On recognition, the protease enzyme proteolyses the protease recognition site, thereby freeing the extracellular domain 6 of the recombinant protein from the rest of the recombinant protein. The cleaved extracellular domain 6 of the recombinant protein is released into the growth medium in the next step 15 of the method 10. In the next step 16 of the method 10, the cleaved extracellu^¬ lar domain 6 of the recombinant protein is harvested from the culture medium and purified. The culture medium containing the extracellular domain 6 of the recombinant protein is transferred to a column 9 comprising affinity silica beads. The extracellular domain 6 of the recombinant protein binds to the affinity silica beads while the remaining culture me^¬ dium flows out. The extracellular domain 6 of the recombinant protein bound to the affinity silica beads in the column 9 can be eluted out of the column using a suitable buffer.

FIG 2 illustrates an embodiment of an expression vector 1 for obtaining one or more domains of protein in their native state. The expression vector 1 comprises of a promoter se^¬ quence 20, a selectable marker gene 23, two multiple cloning sites 21,22, and a termination sequence 24. In the embodi^¬ ment, the selectable marker gene 23 is Geneticin, allowing for positive selection of transfected cells. Geneticin blocks polypeptide synthesis, therefore inhibiting protein chain elongation. Alternatively, other selectable markers known to a person skilled in the art may be used in the expression vector 1. The multiple cloning site 22 is present within the Geneticin marker gene 23 and a part of the gene for the re^¬ combinant protein 2 would be inserted into the multiple clon- ing site 22. Therefore, the cells which have been transfected with the expression vector 1 containing the gene for recombi^¬ nant protein 2 will continue to synthesize proteins, whereas the cells receiving the expression vector 1 that does not have the gene for recombinant protein will not synthesize proteins due to expression of Geneticin. The transcription of the expression vector 1 is terminated with the termination sequence 24.

In the embodiment described in FIG 2, a gene for protease recognition site 3 is present between two multiple cloning sites 21, 22. Therefore, the gene for the membrane domain of the recombinant protein 2 may be cloned in the multiple clon^¬ ing site 21, upstream of the gene for protease recognition site 3 and the gene for extracellular domain of the recombi^¬ nant protein may be cloned in the multiple cloning site 23, downstream of the gene for protease recognition site 3.

Claims

1. A method (10) of producing one or more domains of pro^¬ tein in their native state, said method (10) comprising the steps of:

a. introducing one or more enzyme recognition sites (3) within a gene for a recombinant protein (2), wherein said gene for recombinant protein (2) encodes for said one or more domains of protein;

b. forming an expression construct (4) by cloning said gene for said recombinant protein (2) into an expres^¬ sion vector ( 1 ) ;

c. transferring said expression construct (4) into mam^¬ malian cells (5) ;

d. expressing said gene for recombinant protein (2) con^¬ taining one or more enzyme recognition sites (3) in said mammalian cells (5) ;

e. contacting said expressed recombinant protein with an enzyme specific to said one or more enzyme recogni^¬ tion sites; and

f . producing one or more domains of proteins in their native state.

2. The method (10) according to claim 1, wherein the step of introducing said one or more enzyme recognition sites (3) within said gene for recombinant protein (2) com^¬ prises :

inserting said one or more enzyme recognition sites (3) between gene sequences encoding one or more domains of protein .

3. The method (10) according to claims 1 or 2, wherein: one or more domains of protein in their native state comprises one or more domains of protein in a correctly folded state.

4. The method (10) according to any of the preceding claims, wherein:

said expression construct is transferred into mammalian cells using a transfection process.

5. The method (10) according to any of the preceding

claims, wherein:

said one or more enzyme recognition sites (3) are prote^¬ ase recognition sites.

6. The method (10) according to any of the preceding

claims, wherein:

said enzyme (8) is a protease enzyme.

7. The method (10) according to any of the preceding

claims, wherein

said enzyme (8) a combination of protease enzyme and one or more enzymes, said one or more enzymes are selected from a group consisting of hydrolases, esterases and lyases .

8. The method (10) according to any of the preceding

claims, comprising:

purifying said one or more protein domains of interest of said recombinant protein.

9. The method (10) of claim 8, wherein:

said one or more protein domains of interest of said re^¬ combinant protein is purified using chromatography tech^¬ niques ( 9) .

10. The method (10) according to any of the preceding

claims, wherein:

said gene for recombinant protein (2) is a gene for a recombinant transmembrane protein.

11. The method (10) of claim 10, wherein: said one or more enzyme recognition sites are inserted between gene sequences encoding for membrane domain and extracellular domain of said recombinant protein.

12. An expression vector (1) for use in producing one or more domains of protein in their native state, said ex^¬ pression vector (1) comprising:

a. one or more multiple cloning sites (21,22); and b. one or more gene sequences encoding one or more en^¬ zyme recognition sites (3) , said gene sequences flanked on either side by said one or more multiple cloning sites (21,22).

13. A kit for producing one or more domains of protein in their native state, said kit comprising:

a. one or more single type mammalian cell lines (5); and b. an expression vector (1) with one or more gene se^¬ quences encoding one or more enzyme recognition sites (3) .

14. The kit according to claim 13, further comprising:

an enzyme (8) specific to said one or more enzyme recog^¬ nition sites (3) .

15. The kit according to claims 13 or 14, further compris^¬ ing :

an expression vector with at least one gene for an en^¬ zyme specific to said enzyme recognition sites (3) .

16. The kit according to any of the preceding claims 13 to 15, wherein:

said one or more single type mammalian cell lines (5) constitutively express an enzyme (8) specific to said one or more enzyme recognition sites (3) .