Field of invention:
The invention relates to a fusion tag comprising Serine-aspartic acid repeats of the well
conserved region of the Staphylococcus aureus Sdr C gene superfamily. A START codon
and an enterokinase cleavage site has been incorporated into this repeat region to make a
novel fusion tag that is responsible for expressing soluble proteins in bacterial system.
The invention also involves a kit for expression of soluble proteins. The present invention
also relates to a method of improving the solubility of protein when the protein is
produced in vivo.
Background of the invention:
The advent of recombinant DN A technology and its application has made a number of
recombinant therapeutics available for human use. Prokaryotic or eukaryotic (yeast and
mammalian) expression systems are generally used for recombinant protein production.
Among these, E. coli has been widely used for recombinant protein production. The
system offers high productivity, high growth and production rate, ease of use and
economy. E. coli facilitates protein expression by its relative simplicity, is inexpensive,
fast growth, well-known genetics and the large number of compatible tools available for
biotechnology. Especially the varieties of available plasmids, recombinant fusion
partners and mutant strains have also advanced the possibilities of obtaining
recombinant therapeutics with E.coli system. However, there are a few disadvantages as
lack of post translational modifications, lack of proper secretion system for efficient
release of produced protein into the growth medium, inefficient cleavage of amino
terminus methionine which can result in lower protein stability increased
immunogenicity, limited ability to facilitate extensive disulphide bond formation,
improper folding resulting in inclusion body formation.
Inclusion bodies produced in E.coli are composed of densely packed denatured protein
molecules in the form of particles and proteins residing in inclusion bodies are often
inactive. In order to get an active protein, optimization of the expression conditions or
the refolding studies are required which could be time consuming and cost intensive.

On the other hand, many mammalian proteins can not be expressed successfully in E.
coli which leaves researchers either to explore expression in a wide range of organisms
like baculovirus expression system, gram positive organisms, Pseudomonas expression
system and E. coli hosts at different temperatures along with various fusion tags.
Since insoluble proteins expressed in E. coli hosts require, complicated in vitro
renaturation step and is indeed a low efficient process and even complex for proteins
with multiple disulphide bonds, there is always a necessity for production of soluble
recombinant protein in E. coli as the purification of highly expressed soluble protein is
less expensive and time consuming than refolding and purification from inclusion
bodies. Soluble protein production in E. coli is still a major bottleneck for researcher
and many attempts have been undertaken to improve the solubility or folding of
recombinant protein produced in E. coli. Of various strategies, co-expression of
chaperone proteins such as E.coli GroEs, GroEl, DnaK and DnaJ, lowering incubation
temperature, use of weak promoters, addition of sucrose and betaine in growth medium,
use of richer medium with phosphate buffer such as TB, translocation to periplasm,
fermentation at extreme pH, and use of fusion tags are examples of a few approaches.
Also, proteolytic degradation of recombinant proteins represents a major problem related
to production of gene products in heterologous hosts. Several alternative strategies for
stabilization of expressed gene products are available many of which often give dramatic
stabilization effects. Optimization of fermentation conditions or downstream processing
schemes together with these strategies is solutions to these problems. Various genetic
approaches to improve the stability of recombinant proteins include (i) choice of host cell
strain, (ii) product localization, (iii) use of gene fusion partners, and (iv) product
engineering. In addition, the solubility of the gene product can be influenced by factors
such as growth temperature, promoter strength, fusion partners, and site-directed changes.
Altogether, a battery of approaches can be used to obtain stable gene products.
One of the best approaches to deal with solubility and stability has been to express
proteins as N- or C- terminus fusions. Prior art show that formation of secondary
structures in transcribed mRNA reduces expression of heterologous genes. These

secondary structures interfere with the binding of ribosome with mRNA thereby prevent
efficient translation initiation. These deleterious secondary structures more likely occur
due to short-range RNA-RNA interactions. Sequence determinants at both N- and C-
termini of proteins can influence their stability towards protease degradation. Although
various alterations of expression conditions can sometimes solve the problem, the best
available tools to date have been fusion tags that enhance the solubility of expressed
proteins. However, a utility of these solubility fusions has been difficult since many
proteins react differently to the presence of different solubility tags with some tags
resulting in incorrect folding and some causing inactivity of some proteins.
Proteins do not naturally lend themselves to high-throughput analysis because of their
diverse physiochemical properties. Consequently, affinity tags have become
indispensable tools for structural and functional proteomics initiatives. Affinity tags are
highly efficient tools for protein purification. They allow the purification of virtually
any protein without any requirement of any prior knowledge of its biochemical
properties. Though originally developed to facilitate the detection and purification of
recombinant proteins, in recent years the fusion tag has become clear that affinity tags
can have a positive impact on the yield, solubility and even the folding of their fusion
partners. However, no single affinity tag is optimal with respect to all of these
parameters; each has its strengths and weaknesses. Therefore, combinatorial tagging
might be the only way to harness the full potential of affinity tags in a high-throughput
setting.
There are several fusion tags available for the ease of expression and purification of
recombinant proteins and the smallest fusion tag available is His-tag (6-10 aa). This has
potential problems of leakage of Ni2+ ions used during for purification of His-tag
proteins. The other tags available are thioredoxin (109aa), Glutathione S-transferase
(236aa), maltose binding protein (363aa), NusA (435 aa) etc. Most of these tags are
affinity tags are large in size and mostly they facilitate purification of the fused protein.
Some of them are (thioredoxin, NusA etc) also reported to increase the solubility of the
target proteins compared to unfused proteins when over expressed. Therefore, all the
above-mentioned fusion tags are either affinity tags or they offer solubility. The advent

of high-throughput structural genomics programs and advances in cloning and
expression technology afford us a new way to compare the effectiveness of solubility
tags and the use of affinity tags has therefore become widespread in several areas of
research e.g., high throughput expression studies aimed at finding a biological function
to large numbers of yet uncharacterized proteins.
US2006/0234222 discloses method of producing a soluble bioactive domain of a
protein, the method comprising the step of selecting suitable soluble subunits of a
protein and assessing the produced protein for desired activity. The method may
comprise the steps of amplifying DNA encoding at least one candidate soluble domain,
cloning the amplified DNA into at least one expression vector, using each of said
vectors into which the DNA has been cloned to each transfect or transform one or more
host cell strains, expressing said DNA in one or more host cell strains, and analyzing
expression products from said host cells for solubility.
US6861403 discloses method for expressing proteins as a fusion chimera with a domain
of p26 or alpha crystalline type proteins to improve the protein stability and solubility
when over expressed in bacteria such as E. coli is provided. Genes of interest are cloned
into the multiple cloning site of the Vector System just downstream of the p26 or alpha
crystalline type protein and a thrombin cleavage site. Protein expression is driven by a
strong bacterial promoter (Tac). The expression is induced by the addition of 1 mM
IPTG that overcomes the lac repression (lac Lq). The soluble recombinant protein is
purified using a fusion tag.
US6613548 relates to fusion products prepared by recombinant DNA procedures. The
products are comprised of a soluble protein of interest and an insoluble proteinaceous
tag.
Thus it is known that protein solubility is one of the major problems associated with
over expressing proteins in bacterial system. Protein solubility is judged empirically by
assaying the levels of recombinant protein in the supernatant and pellet of lysed cell
extract. In general proteins with more hydrophilic residues can be found in soluble
fractions of bacterial extracts. In contrast proteins rich in hydrophobic residues or

proteins having complex secondary or tertiary structures are typically insoluble and are
found in inclusion bodies. While in the form of inclusion bodies, the protein will have
no biological activity and will be impossible to purify using affinity fusion tags. These
inclusion bodies can be re-solubilised in chaotropic buffers such as 8M urea or 6M
guanidine hydrochloride, but then must be slowly dialyzed against physiological buffers
in an effort to refold and regain biological function. Due to the individual characteristics
of each protein, this is a slow and painstaking process that may never produce active or
useful protein. Therefore, the ability to quickly produce and screen soluble protein in
bacteria such as E. coli represents a major step forward in protein biochemistry.
Thus the present invention aims at solving the problems of insoluble protein production
by using a fusion tag, the fusion tag comprising Serine-aspartic acid repeat region of
Staphylococcus aureus SdrC gene superfamily with a START codon and an enterokinase
cleavage site to improve the solubility of those proteins which express as insoluble
proteins. Further presence of affinity tags with this fusion tag of present invention would
provide ease of purification.
Objectives of the present invention:
The object of the present invention is a fusion tag comprising Serine-aspartic acid repeat
region of Staphylococcus aureus SdrC gene superfamily with a START codon and an
enterokinase cleavage site.
Another object of the present invention is the use of fusion tag comprising Serine-aspartic
acid repeat region of Staphylococcus aureus SdrC gene superfamily with a START codon
and an enterokinase cleavage site to increase the solubility of proteins.
Another object of the present invention is a vector comprising fusion tag comprising
Serine-aspartic acid repeat region of Staphylococcus aureus SdrC gene superfamily with
a START codon and an enterokinase cleavage site and additional amino acids at the N
terminal region of the serine aspartic acid repeat units

Another object of the present invention is a kit for expression of soluble proteins
comprising vector comprising a Fusion tag comprising of additional aminoacids at the N
terminal region and the SD repeat region of Staphylococcus aureus SdrC gene
superfamily with a START codon and an enterokinase cleavage site actually offers the
solubility factor for the gene of interest.
Another object of the present invention involves a method for producing soluble and
active recombinant protein comprising: (a) cloning fusion tag comprising SD repeats in
the vector (b) cloning additional amino acid sequence in step (a) (c) introduction of gene
of interest in step (b) (d) Transformation of vector from step (d) in E Coll (e) expression
of fusion protein (f) Separation of protein of interest from fusion protein.
Brief Description of The Accompanying Drawings
Figure 1: Colony PCR with T7 forward and GM reverse primers.
Figure 2; Xbal/SnaBI digestion of the pCGMSD construct
Figure 3: Colony PCR with T7 reverse and forward primer
Figure 4: Clone map for GMSD-GCSF
Figure 5: SDS-PAGE profile of expressed GMSD-hGCSF
Figure 6: Clone map for GMSD-hlLl 1
Figure 7: Clone map for GMSD-hIL2
Figure 8: Clone map for GMSD-Reteplase
Figure 9 : SDS-PAGE profile of expressed GMSD-hlLl 1
Figure 10: SDS-PAGE profile of expressed GMSD-hIL2, enterokinase and Reteplase
Detailed description of the invention:
The present invention provides a method for improving the solubility of target protein
when the target protein is produced in bacteria
Another embodiment of the present invention provides a method for expressing target
protein using a vector comprising a fusion tag, comprising serine-aspartic acid (SD)
repeat region of SdrC protein family along with gene of interest with additional amino

acids, about 10 to about 300amino acids at the N terminal region. The additional amino
acids may be either derived from vector sequences from MCS or the sequences could be
from extraneous polypeptides that aid in hyper expression of proteins. The additional
amino acids could be used for affinity purification, antibody detection also. The vector
when introduced in E coli would express soluble proteins.
As used herein, the term "tagging" refers to introducing by recombinant methods one or
more nucleotide sequences encoding a peptide tag into a polypeptide encoding gene.
"Fusion protein" refers to the protein whose N terminus is formed by the fusion tag
comprising the C terminus portion of human GM CSF and a non GM peptide at the C
terminus.
The fusion protein must be continuous with the target protein. The same open reading
frame of the target protein must be maintained with respect to the open reading frame of
the fusion tag. Stop codons between the target protein and the fusion partner must be
omitted.
Vectors suitable to be used for the present invention are numerous and a list of the
vectors can be found in the art. The vectors commercially available from Stratagene,
Promega, CLONTECH, Invitrogen GIBCO Life Sciences and other companies making
expression vectors. All the vectors with bacterial promoters may be used.
Vectors particularly suitable are plasmid vectors, which include prokaryotic, eukaryotic
and viral sequences. A list of these vectors can be found in Gene Transfer and Gene
Expression: A Laboratory Manual, Ed. Kriegler, M., Stockton Press, New York (1990)
and Molecular Cloning, A Laboratory Manual, CSH Laboratory Press, Cold Spring
Harbor, N.Y. and Current Protocols in Molecular Biology, Vol. 1, Supplement 29,
section 9.66, Ed. Asubel, F. M. et al., John Wiley & Sons (2001).
The present invention involves a fusion tag comprising the serine-aspartic acid repeat
(SD) region of SdrC protein family of a gram positive bacterium. Staphylococcus aureus.
Another embodiment of the present invention involves a fusion tag comprising serine-
aspartic acid repeat (SD) region of SdrC protein family of a gram positive bacterium,

Staphylococcus aureus which comprises of 55 each of serine and aspartate residues along
with additional amino acids, about 10 to about 300amino acids at the N terminal region.
The additional amino acids may be either derived from vector sequences from MCS or
the sequences could be from extraneous polypeptides that aid in hyper expression of
proteins. The additional amino acids could be used for affinity purification, antibody
detection also. The additional amino acid sequence may be any which is known in the art
such as GST tag. His tag, T7 tag Trx tag, MBP tag etc.
The most preferable is a 45 amino-acid long peptide and is the C-terminus part of human
Granulocyte Macrophage Colony Stimulating Factor (hGMCSF) gene product. hGMCSF
is a glycoprotein growth factor that induces proliferation of hematopoetic proginator .
The processed hGMCSF polypeptide is 127 amino acid long and of molecular mass of
14.36 kDa. This tag is small and hence upon expression, the molar ratio of the gene of
interest would be highest for a tag which is the smallest in size since the other well
known tags are very large in size.
There are three members of the cell surface-associated serine-aspartate family of proteins
in S. epidermidis, namely, SdrF, SdrG (Fbe), and SdrH, and they are all characterized by
the distinctive serine-aspartate dipeptide (SD) repeats. The overall structure of the coding
region was found to follow the general pattern observed in other Sdr family proteins and
included a signal sequence, an A domain, a repetitive domain termed BX, an SD repeat
region, a cell wall anchor region with an LPXTG motif sequence (LPDTG, amino acids
674 to 678), a hydrophobic membrane-spanning region, and a series of positively charged
residues at the C terminus.
Serine-aspartate repeats have previously been shown to allow a high degree of
discrimination in S. aureus. Initial surveys revealed the largest amount of size variation in
sdrG PCR amplicons, and the gene was present in all strains surveyed.
There were three differently sized PCR amplicons of the SD repeat region from the 48
strains analyzed (-200 bp, ~4 to 500 bp, and ~8 to 900 bp), and there was 100%
concordance between the size of the PCR fragment and the number of repeat cassettes.

The DNA sequence revealed 69 alleles of the repeat cassette, composed of 1 21-bp, 4 12-
bp, and 64 different 18-bp repeats .The SD repeats had earlier been found in the S. aureus
fibrinogen-binding clumping factors ClfA and ClfB. The clfA and clfB genes encode
high-molecular-mass fibrinogen-binding proteins that are anchored to the cell surface of
S. aureus .
SdrC family of proteins are membrane bound protein and consists of several functional
domains. The C termini contain LPXTG motifs and hydrophobic amino acid segments
characteristic of surface proteins covalently anchored to peptidoglycan . The fibrinogen-
binding clumping factor protein of S. aureus is distinguished by the presence of a serine-
aspartate (SD) dipeptide-repeat region. These Sd repeats span the cell wall and extend the
ligand binding region from the surface of the bacteria and sdrC gene is abundant as a
surface protein in several staphylococcus strains. Thus these SD-repeat regions would
most probably enhance the solubility and promote the proper folding of its fusion
partners in E. coli. Also, both the serine and aspartic acid are polar amino acids and has a
high solubility offering solubility of otherwise insoluble proteins.
One of the embodiments of the present invention involves the method of producing
soluble protein the method comprising (a) cloning of SD repeats in the vector (b) cloning
of additional aminoacids in the N terminal region of SD repeat units in step (a) (c)
introduction of gene of interest in step (b) (d) Transformation of vector from step (d) in E
Coli (e) expression of fusion protein (f) Separation of protein of interest from fusion
protein.
The present invention also involves a kit comprising a vector comprising a fusion tag
comprising Serine aspartic acid repeat units. The kit may be used for providing soluble
and active protein of interest.
Description of a preferred embodiment of the present invention:
Example 1: Construction of fusion tag vector
The serine-aspartate (SD) repeat region was synthesized as a synthetic DNA and cloned
into a commercial vector utilizing T7 promoter based vector namely pET21a vector. The

SD stretch fragment was released from the synthetic DNA as an Ndel/EcoRl fragment
and cloned into pET21a at the same sites. Nucleotide sequence corresponding to the
enterokinase cleavage site was incorporated between BamHI and EcoRI sites in the SD
repeat.
The additional amino acids at the N terminal region of the SD repeat units may be GST
tag, His tag, T 7 tag Trx tag, MBP tag etc.
For the present example GM tag is used. The tag is small and hence upon expression, the
molar ratio of the gene of interest would be highest for a tag which is the smallest in size
since the other well known tags are very large in size.
GM tag (the C-terminus domain of hGMCSF) was amplified from a full length human
GM-CSF synthetic gene using gene specific primers
SEQUENCE ID 1:
Forward primer: 5' ccg ccg gaa ttc cat atg cac tac aag cag cac tgc cct cca 3'
SEQUENCE ID 2:
Reverse primer: 5' ccg ccg gaa ttc ttt ate ate ate ate gga tec gac tgg etc cca gca gtc 3'
PCR was performed in a total volume of 250 ul containing 100 pg of a synthetic gene
(Gene bank accession no. BCl08724), 3U of Taq DNA polymerase, 200uM dNTPs
(Bangalore Genei Pvt. Ltd. India) and 10pmoles each of primers (Sigma). Amplification
was done in a two step manner at 94 °C for 5 min followed by 5 cycles of 94 °C for 30 s,
50°C for 30 s and 72 °C for 30 s; 25 cycles of 94 °C for 30 s, 62 °C for 30 s and 72 °C for
30 s and final primer extension at 72 °C for 5 min. The PCR product was digested with
NdeI and cloned into pET21a vector (Novagen) as Ndel fragment. The constructed vector
was designated as pCGMSD and the enterokinase (EK) cleavage site was introduced into
the vector to obtain target protein with no extra amino acids at the N-terminus. Thus the
fusion tag vector, pCGMSD was constructed by cloning GM tag and SD repeat into E.
coli expression vector pET21a. The incorporation of GM was verified by colony PCR
screening with T7 promoter primer and GM reverse primers. Figure 1 indicates colonies
showed PCR product corresponding to GM tag. The incorporation of GM tag was further
verified by restriction digestion with XbaI/SnaBI (Figure 2).

Example 2: Cloning of human Granulocyte Colony Stimulating Factor (hGCSF) in
pCGMSD
hGCSF was amplified from a synthetic gene using gene specific primers
SEQUENCE ID 3:
Forward: 5' CCG CCG GGA TCC GAT GAT GAT GAl AAA ACG CCA TTA GGC
CCG GCC 3'
SEQUENCE ID 4:
Reverse: 5' CCG CCG GAA TTC AAG CCT TAA CGG CTC CGC TAA ATG ACG
3'. PCR was performed in a total volume of 250ul containing 100 pg of synthetic gene
(Gene bank accession no. DQ914891), 3U of Taq DNA polymerase, 200UM dNTPs and
1 Opmoles each of primers. Amplification was done in a two step manner at 94 °C for 5
min followed by 30 cycles of 94 °C for 30 s, 63 °C for 30 s and 72 °C for 30 s and final
primer extension at 72 °C for 5 min. The PCR product was digested with BamHI/EcoRl
and cloned into pCGMSD as BamHI/EcoRI fragment. Clones were screened by colony
PCR (figure 3) and the construct was designated by pCGMSD-hGCSF (Figure 4).
Example 3: Expression of GMSD-hGCSF fusion protein in E. coli host BL21(DE3).
The pCGMSD-hGCSF construct was introduced into E. coli expression host BL21 (DE3)
by a method known as transformation. The cells were induced with ImM IPTG and
induction was carried out for 4 hours as described before. The sub cellular fractionation
was done after cell lysis and soluble and insoluble fractions were separated, analysed on
SDS-PAGE. Figure 5 shows more than 80% hGCSF protein was residing in the soluble
fi-action indicating that GMSD fusion indeed offers solubility to hGCSF. Introduction of
enterokinase cleavage site between fusion tag and target protein helps in obtaining target
proteins with no extra amino acid at its amino terminus.
Example 4: Immunoblot with anti-hGMCSF antibody and Purification of GMSD
tag fusion proteins
GM fusion proteins could be detected and quantified by immunoblot or ELISA with
commercially available anti-hGMCSF antibody. Human GCSF was cloned in pCGMSD
vector and expressed in BL21(DE3) E. coli host. Immunoblot analysis was carried out

with both mouse anti-hGCSF and rabbit anti-hGMCSF antibodies. GM-GCSF fusion
protein is detected by both GCSF as well as GMCSF antibodies. As expected, untagged
GCSF is detected only by GCSF antibody and not by GMCSF antibody.
The fusion tag has an affinity to bind to heparin [Sebollela et. al.. Journal of Biological
Chemistry 280 31049-31956; 2005] and thus can be purified by affinity chromatography
using immobilized heparin sepharose matrices. Human IL11 expressed as GM fusion,
was allowed to bind to heparin sepharose affinity column at pH 5 and eluted at alkaline
pH with buffer containing high salt, IL 11 was found to be purified and fully biologically
active.
Example 5: Biological activity of the fusion protein
NFS60 cell proliferation assay was carried out to check the biological activity of hGCSF
with fusion tag and it has been found to be active in tagged protein.
Example 6: Construction of a fusion tag vector and cloning of human tissue
plasminogen activator (reteplase), Interleukin-2, enterokinase and Interleukin-11
genes in pCGMSD vector
All the above gene products have been reported to occur as insoluble inclusion bodies in
E. coli system. All these genes were cloned as BamHI/HindIII into a vector containing
GM-SD tag under pET21a vector (Figs. 6, 7, 8).
All the genes were screened using gene specific PCR and then clones were screened for
expression for fusion proteins of GM-SD-Reteplase, GM-SD-IL-2, GM-SD-IL-11 and
GM-SD-enterokinase (EK) in BL21(DE3) cells using 1 mM IPTG as the inducer.
The results indicate expression of the fusion proteins as soluble entities as evident from
Figures 9 and 10.
Industrial Applicability
• Improved solubility (S) - Fusion of the N-terminus of the target protein to the C-
terminus of a soluble fusion partner often improves the solubility of the target
protein.

• Improved detection (D) - Fusion of the target protein to either terminus of a short
peptide (epitope tag) or protein which is recognized by an antibody (Western blot
analysis) or by biophysical methods (e.g. GFP by fluorescence) facilitates the
detection of the resulting protein during expression or purification.
• Improved purification (P) - Simple purification schemes have been developed for
proteins used at either terminus which bind specifically to affinity resins.
• Localization (L) - Tag, usually located on N-terminus of the target protein, which
acts as address for sending protein to a specific cellular compartment.
• Improved Expression (E) - Fusion of the N-terminus of the target protein to the C-
terminus of a highly expressed fusion partner results in high-level expression of
the target protein.
• The tag provide for fusion to a polypeptide that itself is highly soluble (e.g. GST,
Trx, NusA)
• provide for fusion to an enzyme that catalyzes disulfide bond formation (e.g.
thioredoxin, DsbA, DsbC)
• provide a signal sequence for translocation into the periplasmic space
• Proteins, which are prone to insoluble aggregates due to higher content of
cysteines, could be easily made soluble using this novel fusion tag.
• This provides a cost effective and time saving way of preparation of soluble
proteins
• This tag could also prove useful for several eukaryotic proteins, which are prone
to go to inclusion bodies in E. coli.

We claim:
1. Fusion tag comprising Serine-aspartic acid (SD) repeat region of Staphylococcus
aureus SdrC gene superfamily with a START codon and an enterokinase cleavage
site.
2. The fusion tag as claimed in claim 1 comprising 107 amino acids of Serine-
aspartic acid repeat region of Staphylococcus aureus SdrC gene superfamily.
3. The fusion tag as claimed in any of claims lor 2 comprising nucleotide sequence
ID 7
ATGAGCGATTCCGATTCAGACTCGGACTCGGATTCCGATTCCGACAGTGA
TTCAGATTCTGACTCAGATTCCGATTCTGATTCTGATTCGGATTCCGACTC
CGATAGCGACTCAGATAGTGACTCTGACTCGGACAGCGATTCTGATAGCG
ACTCTGATTCCGATAGCGATAGCGATTCAGATAGCGATTCTGACTCGGAT
TCTGATTCCGATTCTGACTCTGACAGCGATTCCGATAGCGACAGCGACTCT
GATAGTGATTCAGACTCTGATTCTGATAGTGATAGCGATTCGGATAGTGG
ATCCGATGATGATGATAAA
4. The fusion tag as claimed in any of claims lor 2 comprising amino acid sequence
ID 8 as under:
MSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDS
DSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSDSGSDD
DDK
wherein D D D D K is the enterokinase cleavage site at the carboxy end of the
construct.
5. The fusion tag as claimed in 1 further comprising additional amino acid selected
from T7tag, GST tag, His tag, Trx tag, MBP tag, GM tag
6. The fusion tag as claimed in 5 wherein the additional amino acid which is GM
tag.

7. The fusion tag comprising Serine-aspartic acid repeat region of Staphylococcus
aureus SdrC gene superfamiiy with a START codon and an enterokinase cleavage
site adapted to increase the solubility of proteins.
8. The fusion tag as claimed in 7 further comprising additional amino acid selected
fromT7tag, GST tag, His tag, Trx tag, MBP tag, GM tag.
9. The fusion tag as claimed in 8 wherein the additional amino acid is GM tag.
10. A vector comprising fusion tag having a Serine-aspartic acid repeat region of
Staphylococcus aureus SdrC gene superfamiiy with a START codon and an
enterokinase cleavage site.
11. The vector as claimed in 10 wherein the fusion tag further comprise of additional
amino acid selected from T7tag, GST tag, His tag, Trx tag, MBP tag, GM tag
12. The vector as claimed in 11 wherein additional amino acid in the fusion tag is GM
tag.
13. A kit for expression of soluble proteins comprising vector comprising a fusion
tag comprising Serine-aspartic acid repeat region of Staphylococcus aureus SdrC
gene superfamiiy with a START codon and an enterokinase cleavage site and
addition amino acids.
14. The kit as claimed in 13 wherein the fusion tag further comprise of additional
amino acid selected from T7tag, GST tag. His tag, Trx tag, MBP tag, GM tag.
15. The kit as claimed in 14 wherein additional amino acid in the fusion tag is GM
tag.
16. A method for producing soluble and active recombinant protein comprising: (a)
cloning fusion tag comprising SD repeats in the vector (b) cloning additional
amino acid sequence in step (a) (c) introduction of gene of interest in step (b) (d)

Transformation of vector from step (d) in E Coli (e) expression of fusion protein
(f) Separation of protein of interest from fusion protein.
17. A method of claim 16 wherein SD repeats has the ability of improving the
solubility of protein of interest.
18. The method as claimed in 16 wherein the fusion tag further comprises additional
amino acid selected from T7tag, GST tag. His tag, Trx tag, MBP tag, GM tag
19. The method as claimed in 18 wherein additional amino acid in the fusion tag is
GM tag.

The invention relates to a fusion tag comprising Serine-aspartic acid repeats of the well
conserved region of the Staphylococcus aureus Sdr C gene superfamily. A START
codon and an enterokinase cleavage site has been incorporated into this repeat region
to make a novel fusion tag that is responsible for expressing soluble proteins in bacterial
system.