MODIFIED SAK GENE FOR THE PRODUCTION OF
RECOMBINANT PROTEINS
Field of the invention
The present invention relates to a cloning and expressing modified SAK gene fusion protein
which imparts improved stability to the heterologous protein of interest. Further the invention
relates to process of purification of recombinant heterologous proteins from bacterial
inclusion bodies using modified Staphylokinase protein carrying internal EK site.
Background of the Invention
Fusion proteins are proteins created through the joining of two or more genes which
originally code for separate proteins. Translation of this fusion gene results in a single
polypeptide with functional properties derived from each of the original proteins.
Recombinant fusion proteins are created artificially by recombinant DNA technology for use
in biological research or therapeutics.
Several techniques are available for producing fusion proteins which retain the desirable
characteristics of thermostability, solubility and a high level of expression.
The most commonly used method for producing fusion proteins is use of fusion tags.
Examples of popular fusion tags include, Histidine-tag, glutathione-s-transferase (GST),
Maltose binding protein, NusA, thioredoxin (TRX), polyhistidine (HIS), small ubiquitin-like
modifier (SUMO) and ubiquitin (Ub).
One of the strategies provides a method to express protein of interest as a staphylokinase
(SAK) fusion. Since one can easily assay SAK activity using the simple chromogenic assay,
one could adopt the SAK assay as a measure of successful refolding of the SAK fusion
protein. SAK is a 136 amino acid long bacteriophage encoded protein of 15.5-kDa size and is
devoid of disulphide linkages. SAK is presently undergoing clinical trials for blood clot-lysis
in the treatment of thrombovascular disorders due to its ability to convert plasminogen, (an
inactive proenzyme of the fibrinolytic system) into plasmin, which is a protease. SAK has
gained importance as a potential therapeutic thrombolytic protein and is an extracellular
protein produced by Staphylococcus aureus strains. It is also produced by S. lyicus, S.
simulans, S. seweri and S. xylosus. Schlott et al 273(35): 22346-50, 1998, have disclosed that
SAK is not an enzyme, but rather a cofactor; it forms a 1:1 stoichiometric complex with
plasmin-(ogen) that converts other plasminogen molecules to plasmin, a potent enzyme that
degrades proteins of the extracellular matrix. The high affinity of the SAK-plasminogen
complex for fibrin makes it a promising thrombolytic agent.
Jackson and Tang, Biochemistry, 21(26): 6620-5, 1982 have reported that SAK has been
shown to be homologous to serine proteases although it does not have any protease activity of
its own. Sakharav et al J . Biol. Chem. 271: 27912-27918, 1996, have reported that SAK
structurally resembles plasminogen activators, has plasminogen-binding site and serine
protease domain but does not show protease activity.
IN/1813/KOL/2008 describes that SAK has proteolytic activity, Salunkhe et al 1(1): 5-10,
2009, have reported that the expression levels of SAK-IFN were found to be two folds higher
than that observed with untagged IFN under similar experimental conditions. It has been
observed that a full length SAK (FL-SAK) when expressed results into 2 fragments as mature
SAK and signal peptide. It was found that when FL-SAK was expressed in BL21-A1 cells
resulted into 2 fragments as mature SAK and signal protein. This proves that SAK has
autoproteolytic property when used as C-terminus fusion. Thus, SAK can be used as a
proteolytic tool by exploring its autoproteolytic activity. One of the problems associated with
use of SAK for the expression of protein of interest is that the protein of interest is also
degraded after release of SAK protein from fusion protein.
Summary of the invention
In an aspect the invention is related to fusion protein DNA comprising a first DNA encoding
a modified SAK protein having the nucleotide sequence of SEQ ID No. 1 and a second DNA
fused in the frame encoding the heterologous protein of interest.
In another aspect the invention is related to fusion protein DNA comprising a first DNA
encoding a modified SAK protein having the amino acid of SEQ ID No. 2 and a second DNA
fused in the frame encoding the heterologous protein of interest.
In another aspect the invention relates to a process for the preparation of heterologous protein
in E. coli comprising the steps of:
a) preparing a fusion DNA comprising a first DNA encoding modified SAK protein and a
second DNA fused in the frame encoding the heterologous protein of interest,
b) cloning of the fusion DNA of step a into an expression vector,
c) expressing the fusion protein in E. coli cells,
d) optionally refolding and purifying the fusion protein;
e) enterokinase cleavage of fusion protein, and
f) isolating and purifying the protein of interest to obtain pure heterologous protein.
In yet another aspect the invention provides method for purification of modified SAK fusion
protein wherein, the fusion protein is expressed as inclusion bodies in E. coli comprising the
steps of:
a) solubilizing the inclusion bodies,
b) refolding the solubilizing inclusion bodies,
c) enterokinase digestion of modified SAK fusion protein, and
d) isolating and purifying the protein of interest by one or more of chromatographic
techniques.
In an embodiment of the invention the protein may be refolded before or after enterokinase
digestion.
In another embodiment the fusion protein or the protein of interest can be purified by
purification methods comprising Ion exchange chromatography, affinity chromatography
hydrophobic interaction chromatography, reverse phase chromatography and gel filtration
chromatography
The details of one or more embodiments of the inventions are set forth in the description
below. Other features, objects and advantages of the inventions will be apparent from the
description.
Brief description of the accompanying drawings
Figure 1: Construction of pET21a-SAK-Protein of interest gene expression vector
Figure 2: Modified SAK-PTH expression in inclusion bodies
Figure 3: Staphylokinase activity in SAK-PTH and modified SAK-PTH fusions
DESCRIPTION OF SEQUENCE ID
SEQ ID NO. 1: Nucleic acid sequence of modified SAK
Modified SAK
catatgtcaa gttcattcga caaaggaaaa tataaaaaag gcgatgacgc gagttatttt gaaccaacag gcccgtattt
gatggtaaat gtgactggag ttgatggtaa aggaaatgag ttgctatccc ctcattatgt cgagtttcct attaaacctg
ggactacact tacaaaagaa aaaattgaat acgatgatga tgataaagaa tgggcattag atgcgacagc atataaagag
tttagagtag ttgaattaga tccaagcgca aagatcgaag tcacttatta tgataagaat aagaaaaaag aagaaacgaa
gtctttccct ataacagaaa aaggttttgt tgtcccagat ttatcagagc atattaaaaa ccctggattc aacttaatta
caaaggttgt tatagaaaag aaagatgatg atgataaata a
SEQ ID NO. 2: Amino acid sequence of modified SAK
Modified SAK
Msssfdkgky kkgddasyfe ptgpylmvnv tgvdgkgnel lsphyvefpi kpgttltkek ieylqddddk
yvewaldata ykefrweld psakievtyy dknkkkeetk sfpitekgfv vpdlsehikn pgfnlitkvv iekkgsdddd
k
SEQ ID NO. 3: Nucleic acid sequence of modified SAK-PTH fusion protein
Modified SAK-PTH fusion protein
catatgtcaa gttcattcga caaaggaaaa tataaaaaag gcgatgacgc gagttatttt gaaccaacag gcccgtattt
gatggtaaat gtgactggag ttgatggtaa aggaaatgag ttgctatccc ctcattatgtc gagtttccta ttaaacctgg
gactacactt acaaaagaaa aaattgaata cctgcaggat gatgatgata aatacgtaga atgggcatta gatgcgacag
catataaaga gtttagagta gttgaattag atccaagcgc aaagatcgaa gtcacttatt atgataagaa taagaaaaaa
gaagaaacga agtctttccc tataacagaa aaaggttttg ttgtcccaga tttatcagag catattaaaa accctggatt
caacttaatt acaaaggttg ttatagaaaa gaaaggatcc gatgatgatg ataaatctgt gtccgagatt cagttaatgc
ataaccttgg caaacatttg aactccatgg agcgtgtaga atggctgcgt aagaagttgc aggatgtgca caatttttaa
SEQ ID NO. 4 : Amino acid sequence of modified SAK-PTH fusion protein
Modified SAK-PTH fusion protein
msssfdkgky kkgddasyfe ptgpylmvnv tgvdgkgnel lsphyvefpi kpgttltkek ieylqddddk
yvewaldata ykefrweld psakievtyy dknkkkeetk sfpitekgfv vpdlsehikn pgfnlitkw iekkgsdddd
ksvseiqlmhn lgkhlnsmer vewlrkklqd vhnf
SEQ ID NO. 5: Nucleic acid sequence of modified SAK-IL-1 fusion protein
Modified SAK-IL-1 1 fusion protein
catatgtcaa gttcattcga caaaggaaaa tataaaaaag gcgatgacgc gagttatttt gaaccaacag gcccgtattt
gatggtaaat gtgactggag ttgatggtaa aggaaatgag ttgctatccc ctcattatgt cgagtttcct attaaacctg
ggactacact tacaaaagaa aaaattgaat acctgcagga tgatgatgat aaatacgtag aatgggcatt agatgcgaca
gcatataaag agtttagagt agttgaatta gatccaagcg caaagatcga agtcacttat tatgataaga ataagaaaaa
agaagaaacg aagtctttcc ctataacaga aaaaggtttt gttgtcccag atttatcaga gcatattaaa aaccctggat
tcaacttaat tacaaaggtt gttatagaaa agaaaggatc cgatgatgat gataaagggc caccacctgg cccccctcga
gtttccccag accctcgggcc gagctggaca gcaccgtgct cctgacccgc tctctcctgg cggacacgcg gcagctggct
gcacagctga gggacaaatt cccagctgac ggggaccaca acctggattc cctgcccacc ctggccatga gtgcgggggc
actgggagct ctacagctcc caggtgtgct gacaaggctg cgagcggacc tactgtccta cctgcggcac gtgcagtggc
tgcgccgggc aggtggctct tccctgaaga ccctggagcc cgagctgggc accctgcagg cccgactgga ccggctgctg
cgccggctgc agctcctgat gtcccgcctg gccctgcccc agccaccccc ggacccgccg gcgcccccgc tggcgccccc
ctcctcagcc tgggggggca tcagggccgc ccacgccatc ctgggggggc tgcacctgac acttgactgg gccgtgaggg
gactgctgct gctgaagact cggctgtga
SEQ ID NO. 6: Amino acid sequence of modified SAK-IL-1 1 fusion protein
Modified SAK-IL-1 1 fusion protein
MSSSFDKGKY KKGDDASYFE PTGPYLMVNV TGVDGKGNEL LSPHYVEFPI
KPGTTLTKEK IEYLQDDDDK YVEWALDATA YKEFRVVELD PSAKIEVTYY
DKNKKKEETK SFPITEKGFV VPDLSEHIKN PGFNLITKVV IEKKGSDDDD
KGPPPGPPRV SPDPRAELDS TVLLTRSLLA DTRQLAAQLR DKFPADGDHN
LDSLPTLAMS AGALGALQLP GVLTRLRADL LSYLRHVQWL RRAGGSSLKT
LEPELGTLQA RLDRLLRRLQ LLMSRLALPQ PPPDPPAPPL APPSSAWGGI
RAAHAILGGL HLTLDWAVRG LLLLKTRL
Detailed Description of the invention
According to the present invention, the DNA sequence encoding a heterologous peptide or
protein selected for expression in a recombinant system is fused to a modified SAK DNA
sequence having SEQ ID No. 1 for expression in the host cell. A modified SAK DNA
sequence having SEQ ID NO. 1 is defined herein as a DNA sequence carrying an
enterokinase site in the SnaBI site inside the SAK gene. In modified SAK the
autoproteolytic activity of SAK protein is blocked due to insertion of EK site at 65th aa
position (65th and 69th aa of SAK are reported to be important for complex formation with
plasminogen and induction of active site exposure (Rabijns et al. Nature structural biology,
4(5): 357-360, 1997).
In an embodiment the modified SAK DNA sequence of the invention may be used to express
various proteins of interest as fusion protein of modified SAK protein. A wide variety of
heterologous genes or gene fragments may be used for example but not limited to hormones,
cytokines, growth or inhibitory factors, enzymes, modified or wholly synthetic proteins or
peptides for producing corresponding protein of interest.
The term "protein of interest" as used herein refers to any protein or peptide, the production
of which is desirable. In one embodiment, the protein or peptide is biologically active.
Examples of proteins of interest include, but are not limited to, interleukins i.e. IL-1 1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-15, IL-18, reteplase, parathyroid
hormone(l-34), parathyroid hormone(l-84), thrombopoietin, epidermal growth factor, basic
fibroblast growth factor, granulocyte-macrophage colony stimulating factor, human growth
hormone, granulocyte colony stimulating factor, macrophage colony stimulating factor,
platelet-derived growth factor, interferons including IFN-alpha, IFN-beta, IFN-gamma,
cleavage enzymes such as Factor Xa, thrombin, OmpT and others similar small peptides.
In another embodiment, the protein of interest is human parathyroid hormone PTH (1-34)
and PTH (1-84). Parathyroid hormone is an endocrine hormone synthesized and secreted by
parathyroid gland and regulates calcium and phosphate homeostasis by acting on specific
cells in bone and kidney. Recombinant hPTH is a potent anabolic agent used in the treatment
of osteoporosis. Endogenous PTH is the primary regulator of calcium and phosphate
metabolism in bone and kidney.
Teriparatide (PTH(l-34)) is a truncated portion of full length parathyroid hormone having
amino acid sequence 1 through 34 of the complete molecule comprising 84 amino acids. It
has been approved for the treatment of osteoporosis. Daily injections of PTH(l-34) help to
stimulate new bone formation leading to increased bone mineral density.
Fusion proteins of PTH have been reported with proteins like Streptavidin, Human growth
hormone (Gardella et al J Biol Chem. 265(26): 15854-9, 1990), His-thioredoxin (Liu et
al Protein Express Purification 54:212-219, 2007), in E. coli expression system.
In another embodiment, the protein of interest is interleukins. In a preferred embodiment, the
protein of interest is IL-1 1. IL-1 is a 19 kDa polypeptide consisting of 178 amino acids,
which does not contain potential glycosylation residues, disulphide bonds or other posttranslational
modifications. It binds to a multimeric receptor complex which contains an IL-
11 specific a-receptor subunit and a promiscuous subunit (gpl30). IL1 1 has been
demonstrated to improve platelet recovery after chemotherapy-induced thrombocytopenia,
induces acute phase proteins, modulates antigen-antibody responses, participates in the
regulation of bone cell proliferation and differentiation and could be use as a therapeutic for
osteoporosis. Examples of popular fusion tags for preparation of IL1 1 fusion protein includes
glutathione-s-transferase (GST), Maltose binding protein, NusA, thioredoxin (TRX),
polyhistidine (HIS), small ubiquitin-like modifier (SUMO) and ubiquitin (Ub).
EP1598364A1 discloses novel polynucleotide encoding fusion interleukin (IL)- 11 receptor
and IL-1 1 polypeptide.
In another embodiment there is provided a method of preparing heterologous fusion protein
which further refers to polypeptides and proteins which comprise a protein of interest and a
modified SAK protein. Modified SAK protein carries an enterokinase site inside the SAK
gene. Modified SAK fusion protein is cleaved with enterokinase at two sites one is between
SAK and protein of interest and other is inside the SAK protein. After cleavage of the
modified SAK protein it loses its proteolytic activiy and also staphylokinase activity and
could not degrade the protein of interest further.
In an embodiment different expression vectors may be used for example plasmid, pET or
pBAD, pTOPO or any other expression vector. In a preferred embodiment of the invention
the vector is pET-21a. Though pET-21a contains an inherent ampicillin (antibiotic) marker
for selection of transformants, other antibiotic markers like chloramphenicol acyl transferase
(CAT), Kanamycin (APH, phosphotransferases), tetracycline resistance gene etc. may be
used as antibiotic markers. These markers may be cloned either under a constitutive
promoter or under their own respective promoters at suitable restriction sites of the vector.
Genes of interest are cloned in pET plasmids under control of strong bacteriophage T7
promoter carrying transcription and translation signals is induced by an inducer which
induces expression of T7 RNA polymerase from the host cell which in turn stimulates
expression of protein of interest. T7 RNA polymerase is so selective and active that almost
all of the cell's resources are utilized for gene expression. Vectors carrying genes of interest
are initially propagated in hosts that do not carry T7 RNA polymerase gene in their genomes,
so they are virtually "of and cannot cause plasmid instability due to the production of
proteins potentially toxic to the host cell. Once established, plasmids are transferred into
expression hosts containing a chromosomal copy of the T7 RNA polymerase gene under
lacUV5 control.
The inducible promoters used in the fermentation may be T7 polymerase, uspA or araBAD or
any other promoter present in expression vectors.
The inducer used with the expression construct may be selected from IPTG, lactose,
arabinose or maltose. The use of inducer may be known to person skilled in the art.
Various strains of E. coli may be used for the process of the present invention for example
cells which are protease deficient strains such as BL21, ER2566 and the protease expressing
strains of K12 derivatives such as HB101, JM109, LE392, C600, TOP10, DH5 alpha and the
like.
In preferred embodiment of the invention BL21 (DE3) codon plus competent cells are made
for high level protein expression and easy induction in T7 expression systems. These cells
contain a C01E1 compatible pACYA based plasmid containing extra copies of the argU,
ileY, proL and leuW tRNA genes thus will provide enough tRNA pool for expression of
proteins containing these rare codons for arginine, isoleucine, proline and leucine amino
acids. These cells exhibit antibiotic resistance for chloramphenicol.
In an embodiment the modified cells cloned with SAK-Protein of interest were selected for
fermentation process. The fermentation was carried out in the batch mode using complex
medium comprised of salts like sodium phosphate and potassium phosphate. Inducer used
was IPTG and fermentation batch time was ~ 7 to 8 hours. Growth inhibition was not
observed inspite of inducer addition, this helped to achieve higher biomass along with
expression of protein of interest. This resulted in higher yield of the process
A frequently occurring problem in production of recombinant proteins in Prokaryotic cells is
the formation of hardly soluble intracellular aggregates of denatured forms of protein
expressed called as inclusion bodies, which partially have a secondary structure and can be
found in the cytoplasm of the bacterial cells. The formation of said inclusion bodies leads to
the necessity of solubilizing and renaturing the proteins subsequent to the isolation of the
inclusion bodies by means of centrifugation at moderate speed with the aid of suitable means
in order to maintain their active configuration. Herein, the competitive reaction between a
transfer of the denatured protein into the right folding intermediate and an aggregation of
several protein molecules is an essential factor limiting the yield of renatured protein.
In further embodiment the invention further provides a process for production of pure protein
of interest from the inclusion bodies. For obtaining the pure proteins from the inclusion
bodies one skilled in the art can follow the procedures described in the literature. Typically
the process for the production of pure protein of interest from the inclusion bodies includes
solubilizing the inclusion bodies of proteins; refolding the said solubilized proteins; purifying
the refolded proteins; and isolating pure proteins wherein, the purification step may be
performed more than once before and after the refolding step.
In yet another embodiment the process for production of pure protein of interest from the
inclusion bodies includes solubilizing the inclusion bodies of proteins; refolding the said
solubilized proteins; enterokinase digestion of the refolded protein, purifying the digested
proteins; and isolating pure proteins wherein the steps can be performed in any order.
There are various methods reported in the literature, one skilled in the art can follow one or
more methods to obtain protein of interest. Such techniques have been extensively described
in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,
Volume 152, Academic Press, San Diego, Calif. (1987); Molecular Cloning: A Laboratory
Manual, 2d ed., Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989); Current Protocols in
Molecular Biology, John Wiley & Sons, all Viols., 1989, and periodic updates thereof; New
Protein Techniques: Methods in Molecular Biology, Walker, J . M., ed., Humana Press,
Clifton, N.J., 1988; and Protein Purification: Principles and Practice, 3rd. Ed., Scopes, R. K.,
Springer-Verlag, New York, N.Y., 1987, the above are incorporated herein by references in
its entirety. In general, techniques including, but not limited to, ammonium sulfate
precipitation, centrifugation, ion exchange, reverse-phase chromatography, affinity
chromatography, hydrophobic interaction chromatography may be used to further purify the
protein.
In yet another embodiment, the invention presents a method for purification of fusion protein
expressed as inclusion bodies. The inclusion bodies are solubilized, filtered and refolded by
fast dilution drop wise into refolding buffer followed by diafiltration.
The solubilization buffer is selected from buffer A (lOOmM Tris, 6 M GuHCl, pH 8.0),
buffer B (6M GuHCl, 20 mM Tris-Cl, pH 8.0). The solubilization step comprises
centrifugation or continuous stirring to ensure complete solubilization of the inclusion bodies
in the solubilization buffer. The refolding buffer comprises one or more buffers selected from
buffer A (20 mM Tris, 0.5 M Arginine, 5% Sorbitol, 2 mM EDTA, pH 8.0), buffer B (20
mM Tris, 4 M urea, pH 8.0) and buffer C(20 mM Tris-Cl, pH 8.0, 0.5M Arginine, 5%
Sorbitol and 1 mM EDTA). The solubilization and refolding steps may optionally be
followed by diafiltration.
In another embodiment of the invention, the refolded fusion protein is digested with
enterokinase (Novagen bovine enterokinase) at RT in ImM CaCl2 to release protein of
interest. The enterokinase digestion of the fusion protein is followed by centrifugation and
the supernatant obtained after centrifugation is further used for purification of the protein of
interest.
The protein of interest may be further purified using one or more purification steps. The
purification steps include affinity chromatography, metal affinity chromatography,
hydrophobic interaction chromatography, ion exchange chromatography, Size exclusion
chromatography and others. The sequence of the chromatography may be in any order
depending on the protein of interest and nature of impurities.
The purity of the purified protein is 99% as determined by RP-HPLC. The purified proteins
can further be used for making pharmaceutical compositions.
The invention is further illustrated by the following examples which are provided merely to
be exemplary of the invention and do not limit the scope of the invention. Certain
modifications and equivalents will be apparent to those skilled in the art and are intended to
be included within the scope of the invention.
Example 1: Construction of pET21a-Staphylokinase (SAK) vector:
In-house pET21a-SAK vector was constructed using synthetic staphylokinase (SAK) gene as
a template. SAK gene was amplified using gene specific primers and digested with Ndel -
BamHI enzymes and ligated to pET2 1a vector at the same site.
Also, a pET-modified SAK-gene of interest clone was constructed, after construction of pETSAK-
gene of interest clone. Site directed mutagenesis was performed to insert SnaBI site
into the native SAK gene which is cloned as Ndel/BamHI in pET21a vector. An additional
enterokinase site was then introduced into the SnaBI site by an additional cloning step. Thus
the final pET2 la-modified SAK clone contains two enterokinase sites.
The gene of interest for the desired protein was synthesized with suitable restriction sites at
both the 5' and the 3' end as a synthetic DNA fragment and used as a template for further
cloning experiments. This gene of interest was PCR amplified, purified and digested with
BamHI/Hindlll and cloned in pET-SAK and pET-modified SAK vectors at BamHI/Hindlll
sites and expressed in E. coli BL21(DE3) Codon plus cells as a fusion protein upon
induction in 1mM IPTG.
The basic transformation for the expression studies is by selective induction using a nonmetabolisable
inducer. Briefly, the mixture of a recombinant expression construct namely
pET21a-SAK-gene of interest and pET21a- modified SAK-gene of interest and BL21 (DE3)
Codon plus competent cells were incubated on ice for 30 min individually, followed by heat
shock at 42 °C for 2 min. Post heat shock the cells were placed on ice for 2 min and 800 ΐ of
Luria Bertanni broth (LB) medium was added and incubated additionally for 1 hr at 37 °C.
After 1 hr, the culture (suitable volume) is plated on a LB agar plate containing ampicillin
and chloramphenicol antibiotic at 100 g/ml and 34 g/ml final concentrations of both the
antibiotics respectively. The plates after incubation at 37 °C for 16-18 hr were used for
further selection of transformants. The recombinant clones from both the vectors carrying the
plasmid with the protein of interest gene were chosen for expression studies.
Example 2: Expression of SAK-Protein of interest fusion protein
Expression of the gene of interest is achieved in shake flask studies. Briefly, 50 ml of LB
with amp and chloramphenicol was added and the BL21 (DE3) codon plus cells carrying
either pET21a-SAK- gene of interest and pET21a- modified SAK-gene of interest constructs
were grown at 37 °C till the absorbance of 1.0 at 600 ran. The cultures were induced with 1
mM IPTG for 4 hours and the samples were analyzed on a 15% denaturing polyacrylamide
gels (SDS-PAGE) with suitable negative control (pET21 alone) and suitable protein
molecular weight marker. Finally, the gels were visualized with CBB R-250 staining. After
EK digestion of SAK-Protein of interest fusion at various time points, the protein of interest
was not visible on a suitable Tricine gel. This was thought to be due to the proteolytic
activity of released SAK on the released protein of interest after the EK digestion.
When the modified SAK- gene of interest fusion was digested with EK, the cleavage
occurred at 2 sites one between modified-SAK and the protein of interest and other was
inside the SAK protein. Due to the cleavage of SAK protein, the SAK as a SAK* lost its
proteolytic activity and also the staphylokinase activity and hence could not degrade the
protein of interest further. To test this hypothesis, SAK activity was tested for both the fusion
proteins.
Example 3: Chromogenic assay of SAK
SAK activity was quantified using plasminogen coupled chromogenic assay as described by
Deshpanade et. al. Biotechnol. Lett 31: 8 1-817, 2009. Briefly, 25 mU of human
plasminogen, chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma)
and samples containing SAK were incubated in 100 l reaction volume in 96-well flatbottom
plates (Nunc) at 25 °C for 20 min. Amount of p-nitroaniline (pNA) released was
monitored at 405 nm by plate reader (Multiskan Spectrum, Thermo, USA). One unit (1U) of
SAK is the amount of the enzyme needed to form 1U of plasmin from plasminogen. Units of
plasmin formed were estimated from the amount of chromogen (pNA) formed using a
standard curve of pure pNA.
Figure 3 depicts that SAK-protein of interest fusion has active staphylokinase whereas the
activity is lost in modified SAK-protein of interest fusion.
Modified SAK-protein of interest fusion clone was therefore used in further fermentation
process.
Example 4: Modified SAK-protein of interest fermentation process
Modified SAK-protein of interest fermentation was carried out in the batch mode using
complex medium comprised of salts like sodium phosphate and potassium phosphate.
Glucose, mannitol, sorbitol or glycerol was used as the source of carbon and energy.
Fermentation process parameters followed were that of typical E. coli fermentation process
i.e. pH ~ 6.5 to 7.5, temperature ~ 25 °C to 42 °C. Aeration 0.5 to 2 wm, etc. Inducer used
was IPTG at the concentration ranging from 0.05 mM to 2 mM. Fermentation batch time was
~ 7 to 8 hours, while cell density achieved was OD (6oonm) 60. Growth inhibition was not
observed in spite of inducer addition, this helped to achieve higher biomass along with
expression of protein of interest. This resulted in higher yield of the process. The expression
of modified SAK-protein of interest fusion was 2.5 to 3 g L and was visible in IB.
Depending upon the molar ration of protein of interest and modified SAK in fusion protein,
the yield protein of interest will vary.
Example 5: Purification of PTH from the pET21a-modified SAK-PTH construct
PTH obtained from example 4, as inclusion bodies were further subjected to isolation and
purification steps.
Solubilization of inclusion bodies: The bacterial inclusion bodies of pET21a-modifiedSAKPTH
clone were solubilised in the ratio of 20 ml of solubilization buffer (6M GuHCl, 20 mM
Tris-Cl, pH 8.0) per gm of inclusion bodies. Kept with constant stirring at RT for 30-60
minutes.
Refolding: The solubilised inclusion bodies filtered and refolded by fast dilution drop wise
into refolding buffer (20 mM Tris-Cl, pH 8.0, 0.5M Arginine, 5% Sorbitol and 1mM EDTA)
at the ratio of 1:20. Refolding was kept at 4-8°C with constant stirring for 12-14 hours.
Diafiltration: The refolded sample was concentrated to half the volume and then diafiltered
against 20 mM Tris-Cl, pH 8.0 (2.5-3 diavolumes). The diafiltered sample was then filtered
using 1/0.45 micron filter.
Ek digestion: Digested for 12-14 hrs with enterokinase (Novagen enterokinase) 0.5 units / A
280 of fusion protein at RT in ImM CaC12 to release protein of interest.
Q-Sepharose column: The filtered protein solution was then loaded onto Q-Sepharose
column. Equilibration buffer- 20 mM Tris-Cl, pH 8.0. Elution buffer- 1.0 M NaCl 20 mM
Tris-Cl, pH 8.0. Flow rate: 382 cm/h. The flow through was collected. Elution done with a
step gradient of 100%B. Column volumes used was 9.0-10.0 ml of resin per gm of inclusion
bodies. Volume-250 ml approx for 1 gm inclusion bodies.
SP-Sepharose column: The Q-Sepharose Flow through was adjusted to pH of 6.0 and loaded
on to SP-Sepharose column. Equilibration buffer- 10 mM Tris-Cl, pH 6.8. Elution buffer- 1.0
M NaCl in 10 mM Tris-Cl, pH 6.8. Flow rate: 300 cm/h. Elution done using gradient of 0-
15%B in 12.5 CV. Resin volume used was 18.0 ml per gm of Inclusion bodies at a bed height
of 9.0 cm. Volume- 36 ml per gm of Inclusion bodies.
Phenyl-Sepharose HP column: The SP-Sepharose eluate was adjusted to pH of 4.5 with 1:3
diluted acetic acid and then 1.7 M of ammonium sulphate was added to the protein solution
gradually. Equilibration buffer- 1.7 M ammonium sulphate in 20 mM Sodium acetate, pH
4.5. Elution buffer: 20 mM Sodium acetate, pH 4.5. Flow rate: 150 cm/h. Elution was done
using gradient of 0-50%B in 35 CV. Resin volume used was 9.0 ml per gm of Inclusion
bodies at a bed height of 4.5 cm. the eluates were collected and analyzed by RP-HPLC for
purity. 50-70 ml per gm inclusion bodies.
Gel filtration: The above purified protein was then buffer exchanged against WFI using
Sephadex G10. Loading was at 5% of column volume at a flow rate of 60 cm/h. The purified
protein in WFI was then lyophilized and reconstituted in formulation buffer at the required
concentration.
Example 6: An alternate process for purification of PTH to minimize volume during
refolding using Urea -a different denaturant in place of guanidine hydrochloride.
Downstream Process Development for PTH from SAK*PTH:
Solubilization and refolding of Inclusion bodies: Solubilisation was carried out using 4 M
Urea in 20 mM Tris-Cl, pH 8.0 at room temperature (22 °C-25 °C) for 1-2 hours.
Solubilization was followed by centrifugation at 27000 g for 10 minutes at 10-15 °C.
EK Digestion: Enterokinase digestion was done using Pichia derived human Enterokinase in
the range of 16-20 ng per 1 g of total protein. Digestion was carried out at room
temperature (22°C-25°C) for 14-20 hours.
Q-Sepharose column: The Enterokinase digested protein was filtered using 1.0 micron filter
and loaded on to anion exchange using Q-Sepharose FF at pH 8.0 in 20 mM Tris-Cl. QSepharose
FF is in the Flow Through mode, i.e., the protein of interest , PTH in this case,
comes in the unbound fraction.
SP Sepharose: pH of the Q-Sepharose Flow Thorough was adjusted to pH 4.0 using glacial
acetic acid and was then filtered using 1.0 micron filter. The filtrate was then used as the load
for Cation exchanger, SP-Sepharose FF. The buffer used in this step is 20 mM Sodium
acetate with and without 1.0 M NaCl. Our protein of interest comes out in the eluate. The
conductivity of the above eluate was adjusted to > 150 mS/cm using sodium chloride as the
salt.
Hydrophobic Interaction chromatography: The conductivity adjusted protein in then filtered
and used as the load for Hydrophobic Interaction Chromatography (HIC). Phenyl-Sepharose
HP is used as the HIC resin. Our protein of interest comes out in the eluate.
The above HIC eluate is then readjusted to conductivity > 150 mS/cm using sodium chloride
as the salt and reloaded on Phenyl-Sepharose HP. This second step of HIC is used as a
concentration step.
Sephadex G-10: Finally the protein of interest is loaded on Sephadex G10 (Gel Filtration
Chromatography-GFC) for buffer exchange. The GFC eluate is the final Drug substance
which is stored at 2-8°C.
Example 7: Purification of modified SAK-ILll
IL-1 1 obtained from example 4 as inclusion bodies was further subjected to isolation and
purification steps.
Solubilization of Inclusion bodies: Inclusion bodies were solubilised in the ratio of 20 ml of
solubilization buffer (lOOmM Tris, 6 M GuCl pH 8.0) per gm of inclusion bodies. Spun at
15000 rpm for 15 minutes. The supernatant was used for refolding.
Refolding: The solubilised inclusion bodies was refolded in two different refolding buffer
conditions:
Buffer A: 20 M Tris, 0.5 M Arginine, 5% Sorbitol, 2 mM EDTA, pH 8.0
Buffer B: 20 mM Tris, 4 M urea, pH 8.0
Diafiltration: The refolded sample was concentrated to half the volume and then diafiltered
against lOmM Tris pH 8.0. The diafiltered sample was then filtered using 1/0.45 micron filter
and Spun at 15000 rpm for 15 minutes.
Ek digestion: Digested for 16 hrs with enterokinase (Novagen enterokinase) 2.4 units / 0.4mg
of fusion protein at RT in ImM CaC12 to release protein of interest. After digestion the
protein sample is spun at 15000 rpm, 15min and supernatant is used for purification
CM-Sepharose column: The filtered protein solution was then loaded onto CM-Sepharose
column. Equilibration buffer- 20 mM Tris-Cl, 150 mM glycine, 5mM Methionine pH 8.0.
Elution buffer- 125 M NaCl. Eluted protein is > 95. % pure on HPLC and SDS -PAGE silver
stained gel. The purified protein in WFI was then lyophilized and reconstituted in
formulation buffer at the required concentration. The protein is passed through Q Sepharose
to remove endotoxins. The CM elution is further loaded onto Q Sepharose at 10-1 1 ms
conductivity in 20 mM Tris pH 8.0 to remove endotoxin, the protein comes in the flow
through fraction HPLC profile of Oprelvekin and purified rhIL-1 1.
CLAIMS
1. A fusion DNA comprising a first DNA encoding a modified SAK protein comprising
nucleic acid sequence of SEQ ID NO. 1 and a second DNA fused in frame encoding the
heterologous protein of interest.
2. The fusion DNA as claimed in claim 1, comprising amino acid sequence of SEQ ID NO. 2.
3. The fusion DNA as claimed in claim 1, wherein the heterologous protein of interest is
selected from the group comprising parathyroid hormone (1-34), parathyroid hormone (1-
84), reteplase, interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1 1,GCSF, epidermal growth factor
and platelet derived growth factor.
4. The fusion DNA as claimed in claim 1, wherein the heterologous protein of interest is
parathyroid hormone (1-34) having SEQ ID NO. 3.
5. The fusion DNA as claimed in claim 1, wherein the heterologous protein of interest is
parathyroid hormone (1-34) having SEQ ID NO. 4.
6. The fusion DNA as claimed in claim 1, wherein the heterologous protein of interest is IL-
1 having SEQ ID NO. 5.
7. The fusion DNA as claimed in claim 1, wherein the heterologous protein of interest is IL-
11 having SEQ ID NO. 6.
8. The fusion DNA as claimed in claim 1, wherein the modified SAK protein carries an
enterokinase site inside the SAK gene.
9. A process for the preparation of heterologous protein of interest in E. coli comprising the
steps of:
a) preparing a fusion DNA comprising a first DNA encoding modified SAK protein and a
second DNA fused in the frame encoding the heterologous protein of interest,
b) cloning of the fusion DNA of step a into an expression vector,
c) expressing the fusion protein in E. coli cells,
d) optionally refolding and purifying the fusion protein;
e) enterokinase cleavage of fusion protein, and
f) isolating and purifying the protein of interest to obtain pure heterologous protein.
10. The process as claimed in claim 9, wherein the heterologous protein of interest is
selected from the group comprising cytokines, growth stimulating factors, hormones,
interferons, interleukins, enzymes.
11. The process as claimed in claim 9, wherein the heterologous protein of interest is selected
from the group comprising parathyroid hormone (1-34), parathyroid hormone (1-84),
reteplase, interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1 1, GCSF, epidermal growth factor and
platelet derived growth factor.
12. The process as claimed in claim 9, wherein the heterologous protein of interest is
parathyroid hormone (1-34).
13. The process as claimed in claim 9, wherein the heterologous protein of interest is IL-1 1.
14. A method for purification of modified SAK fusion protein wherein, the fusion protein is
expressed as inclusion bodies in E coli comprising steps of:
a) solubilizing the inclusion bodies,
b) refolding the solubilizing inclusion bodies,
c) enterokinase digestion of modified SAK fusion protein, and
d) isolating and purifying the protein of interest by one or more of chromatographic
techniques.
15. The method as claimed in claim 14, wherein the protein may be refolded before or after
enterokinase digestion.
16. The method as claimed in claim 14, wherein the fusion protein can be purified by
purification methods comprising Ion exchange chromatography, affinity chromatography,
hydrophobic interaction chromatography, reverse phase chromatography and gel filtration
chromatography
17. The method as claimed in 14, wherein ion exchange chromatography is selected from
cation exchange and anion exchange in any order.
18. The method as claimed in 16, wherein hydrophobic interaction chromatography is
performed using resins selected from butyl sepharose, phenyl sepharose or octyl sepharose.