Field of the invention
The present invention relates to a process for producing heterologous proteins of interest
using methionine aminopeptidase (MAP) protein as a fusion tag. Further the invention
relates to fusion protein DNA sequences comprising MAP protein.
Background of the invention
Recent advances in techniques related to recombinant DNA cloning in last two decades has
led to increase in the number of proteins expressed in large scale. These proteins have
therapeutic, diagnostic or industrial importance. The recombinant proteins can be expressed
in variety of different expression systems like bacterial, fungal, yeast and mammalian cell
systems. Each of the above systems has their own advantages and disadvantages. Though
mammalian expression systems will express correctly folded proteins, the overall yields of
protein of interest in this expression system are very low and the cost of production is
relatively highest in comparison to other expression system.
The cloning and expression of foreign proteins in E. coli is highly efficient since E. coli offers
high productivity, high growth and production rate, ease of use and economical. 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.
However, there are a few disadvantages like lack of post-translational modifications, lack of
proper secretion system for efficient release of produced protein into the growth medium,
inefficient cleavage of amino terminal methionine which can result in lower protein stability
and increased immunogenicity, limited ability to facilitate extensive disulphide bond
formation, improper folding resulting in inclusion body formation etc. However, when
heterologous proteins are expressed in E. coli in high levels, recombinant proteins are
frequently expressed in E. coli as insoluble protein aggregates termed as "inclusion bodies".
In general small proteins (>30 kDa) that are simple monomeric proteins can be found in the
soluble fractions of bacterial extracts. In contrast, proteins (<30 kDa) or proteins that have
complex secondary or tertiary structures are typically insoluble and are predominantly found
in inclusion bodies. Although initial purification of inclusion body material is relatively simple,
the protein must be subsequently refolded into an active form which is typically a
cumbersome process. If these additional procedures are not successful then little or no
protein activity is recovered from the host cells. Thus, it is much more desirable to express
the recombinant protein in soluble form.
Several approaches, including protein fusions, chaperone co-expression, and promoter
alterations, have been used to overcome these problems (Zhang Y, Olsen DR, Nguyen KB,

Olson PS, Rhodes ET, Mascarenhas D, 1998, Expression of eukaryotic proteins in soluble
form in Escherichia coli, Protein Expr. Purif. 12, 159-165; Thomas JG, Baneyx F, 1997
Divergent effects of chaperone overexpression and ethanol supplementation on inclusion
body formation in recombinant Escherichia coli, Protein Expr. Purif. 11, 289-296).
Unfortunately, these methods are not widely applicable. One of the strategies to prevent
formation of protein aggregates is to tag the protein of interest with a fusion protein with a
protein known to have been soluble at very high levels. Several commercial fusion protein
tags are available in market like thioredoxin (LaVallie ER, DiBlasio EA, Kovacic S, Grant KL,
Schendel PF, McCoy JM, 1993 A thioredoxin gene fusion expression system that
circumvents inclusion body formation in the E. coli cytoplasm, Biotechnology. 11,187-193.),
Intein, maltose binding protein (Pryor KD, Leiting B1997 High-level expression of soluble
protein in Escherichia coli using a 6X His-tag and maltose-binding-protein double-affinity
fusion system, Protein Expr. Purif. 10, 309-319.), Glutathione S transferase (Nygren PA,
Stahl S, Uhlen M 1994 Engineering proteins to facilitate bioprocessing, Trends Biotechnol.
12,184-188.), SUMO tags (Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR
2006 Comparison of SUMO fusion technology with traditional gene fusion systems:
Enhanced expression and solubility with SUMO, Protein Sci. 15, 182-189.), ZZ tag, NusA
(Marco VD, Stier G, Blandin S, Marco AD 2004 The solubility and stability of recombinant
proteins are increased by their fusion to NusA, Biochem. Biophys. Res. Commun. 322, 766-
771.), Ubiquitin (Catanzariti AM, Soboleva TA, Jans DA, Board PG, Baker RT 2004 An
efficient system for high-level expression and easy purification of authentic recombinant
proteins, Protein Sci. 13,1331-1339.) etc.
Methionine aminopeptidases are ubiquitously distributed in all living organisms. The removal
of the N terminal methionine is a critical step for protein modifications that are important in
controlling protein subcellular localization and/or protein degradation. Two distantly related
MetAP enzymes, type 1 and type 2, are found in eukaryotes, while prokaryotes express only
one type of MAP. MetAP I exit in eubacteria while MetAP II exists in archae ( Li X, Chang YH
1996, Evidence that human homologue of a rat initiation factor -2 associated protein (p67) is
a methionine aminopeptidase Biochem Biophysics Res Comm 227-1, 152-9 ; Dummitt B,
Micka W, Chang YH 2003 N-terminal methionine removal and methionine metabolism is
Saccharomyces cerevisiae J Cell Biochem 89:964-974).
N-terminal methionine removal in bacteria is a two step process requiring the removal of the
N formyl group by polypeptide deformylase first followed by cleavage of the N terminal
methionine when the adjacent amino acid is small. Both the above steps appear to be
essential for bacterial cell viability. Failure to remove the N terminal methionine can lead to
inactive enzymes (e.g. glutamine phosphoribosylpyrophosphate aminotransferase and N
terminal nucleophile hydrolase).

The invention relates to use of MAP protein as a fusion tag to obtain a soluble protein of
interest in E.coli cells.
Summary of the invention
In an aspect the invention relates to a process for the production of the heterologous protein
in E. coli cells, the process comprises of:
a) preparing a fusion DNA comprising a first DNA fragment encoding MAP protein and a
second DNA fragment fused in the frame encoding the heterologous protein of interest,
b) cloning of the vector comprising the fusion DNA of step a,
c) expressing the fusion protein in E. coli cells in soluble form,
d) obtaining protein from the fusion protein and
e) purifying the protein.
In another aspect, the invention is related to a fusion protein DNA sequence comprising
MAP protein sequence fused to a DNA of the heterologous protein of interest.
In another aspect, the invention provides a process for the production of heterologous
protein as a soluble fusion protein using a MAP protein as a fusion tag.
Brief Description of the drawings and SEQ ID.
Figure 1: Expression of the MAP fusion protein in BL 21(DE3) cell line transformed with MAP
protein and purification of MAP protein (Lane 1: Protein Molecular weight marker 14 kDa to
97 kDa; Lane 2: Soluble fraction of pETMAP clone #1; Lane 3: Soluble fraction of pETMAP
clone #1; Lane 4: Elution fraction 200 mM Immidazole #1)
Figure 2: Expression analysis of with different MAP fusion proteins (RTP, IFN, PNGase F,
IL-2, EGF and EK) in transformed BL21 (DE3) cell line (Lane 1: Soluble fraction of
pETMAPRTP clone ; Lane 2: Insoluble fraction of pETMAPRTP clone; Lane 3: Protein
Molecular weight marker 14 kDa to 97 kDa; Lane 4: Protein Molecular weight marker 14 kDa
to 97 kDa ; Lane 5: Soluble fraction of pETMAPPNgase F clone; Lane 6: Insoluble fraction of
pETMAPPNgaseF clone; Lane 7: Protein Molecular weight marker 14 kDa to 97 kDa; Lane
8: Insoluble fraction of pETMAPIL-2 clone ; Lane 9: Soluble fraction of pETMAPIL-2 clone
Lane 10: Protein Molecular weight marker 14 kDa to 97 kDa ; Lane 11: Soluble fraction of
pETMAPIFN clone ; Lane 12: InSoluble fraction of pETMAPIFN clone; Lane 13: Protein
Molecular weight marker 14 kDa to 97 kDa ; Lane 14: Soluble fraction of pETMAPEGF clone

# 1; Lane 15: Insoluble fraction of pETMAPEGF clone # 1 ; Lane 16: Protein Molecular
weight marker 14 kDa to 97 kDa ; Lane 17: Soluble fraction of pETMAPEK clone; Lane 18:
Insoluble fraction of pETMAPEK clone)
Figure 3: Enterokinase digestion of MAP fusion proteins (MAP-RTP, MAP-IFN, MAP-
PNGase F and MAP-IFN fusion) (Lane 1: Protein Molecular weight marker 14 kDa to 97
kDa; Lane 2: Soluble fraction of pETMAPRTP clone ; Lane 3: Soluble fraction of
pETMAPRTP clone digested with enterokinase; Lane 4: Protein Molecular weight marker 14
kDa to 97 kDa ; Lane 5: Soluble fraction of pETMAPIFN clone ; Lane 6: Soluble fraction of
pETMAPIFN clone digested with enterokinase; Lane 7: Protein Molecular weight marker 14
kDa to 97 kDa; Lane 8: Soluble fraction of pETMAPPNgaseF clone; Lane 9: Soluble fraction
of pETMAPPNGaseF clone digested with enterokinase; Lane 10: Protein Molecular weight
marker 14 kDa to 97 kDa ; Lane 11: Soluble fraction of pETMAPEK clone ; Lane 12: Elution
fraction of pETMAPEK soluble fusion after Nickel purification showing self-cleavage activity
of enterokinase in the fusion.)
Figure 4: Schematic representation of vector map of pET21-MapF
Figure 5: Expression analysis of various tested proteins without any tag (Lanes 1, 3, 5, 7:
Protein Molecular weight marker 14 kDa to 97 kDa; Lane 2: Insoluble fraction of E. coli BL21
(DE3) pETRTP clone; Lane 4: Insoluble fraction of E. coli BL21 (DE3) C + pETIFN clone;
Lane 6: Insoluble fraction of E. coli BL21 (DE3) pETEK clone; Lane 8: Insoluble fraction of E.
coli BL21 (DE3) pETIL-2 clone)
SEQ ID NO. 1 : DNA sequence of MAP protein
SEQ ID NO. 2 : Amino acid sequence of MAP protein
Detailed description of the invention
As used herein, "heterologous protein" or "protein of interest" refers generally to peptides
and proteins exogenous i.e. foreign to the E. coli cells. Examples of the protein includes
molecules such as, colony stimulating factors (CSFs), for example M-CSF, GM-CSF, and G-
CSF; growth hormone, including human growth hormone; interferon such as interferon-
alpha, -beta, and -gamma; interleukins (ILs), such as IL-2, IL-11, IL-1RA; reteplase,
staphylokinase, Steptokinase DPP-4, DPP-8, PTH, PDGFAA, PDGFAB, PDGFBB and
fragments of any of the above-listed polypeptides.

In an embodiment of the invention there is provided a fusion DNA comprising MAP protein
and an enzymatic cleavage site. In another embodiment there is provided a fusion DNA
comprising MAP protein and an enzymatic cleavage site wherein the fusion tag increases
the solubility of the protein of interest.
In another embodiment there is provided a vector comprising the fusion DNA comprising
MAP protein an enzymatic cleavage site, protein of interest.
In an embodiment there is provided a process for producing heterologous peptides or
proteins in a soluble and stable form in E. coli cells. Specifically, the invention provides a
method for producing a protein of interest in the soluble form comprising:
a) obtaining a fusion DNA comprising a first DNA encoding MAP protein, Enterokinase site
and a second DNA fused in the frame encoding the heterologous protein of interest,
b) cloning of the vector comprising the fusion DNA of step a,
c) expressing the fusion protein in E. coli cells in soluble form,
d) obtaining protein of interest from the fusion protein and
e) purifying the protein of interest.
In another embodiment any commercial vector and promoter may be used such as pET21a,
pBAD24, pQE, etc. The preferred vector is pET21a.
In an embodiment of the invention any enzymatic cleavage site may be used such as
enterokinase, TEV protease, Factor Xa and thrombin etc. The preferred enzyme cleavage
site is of enterokinase .
The process of the invention provides to express protein of interest as soluble entities in E.
coli cells which are reported to be usually expressed in insoluble form (inclusion bodies). To
obtain the protein of interest from the inclusion bodies, processes like harsh denaturation
conditions, refolding etc are required which are tedious, time-consuming, not universal and
expensive and not applicable for large scale manufacturing scale operations.
In another embodiment the protein of interest which may be expressed by the present
invention are cytokines, hormones, thrombolytic agents, cleavage enzymes, enzymes
related to glycosylation or deglycosylation like PNGaseF, endo HF,, restriction enzymes and
the like. Cytokines includes Interleukins, Interferons, Growth factors, Colony Stimulating
factors and the like. Hormones includes PTH, FSH, GH, LH and the like.
In an embodiment of the invention the present invention may be used to express the protein
of interest which require external supply of rare codons for example interferon or proteins
which have strong secondary mRNA structure for example IL-2.
In an embodiment of the invention MAP protein is used as a fusion tag to express proteins of
interest in E. coli cells is less in size(MAP is 39 kDa) as compared with other tags like NusA

tag, the molar ration of the fusion tag versus protein of interest will be low which in turn
results in higher yield of the proteins of interest after separation from the fusion partner.
In an embodiment of the invention the gene of interest was amplified from a template and
digested with BamHI/Hindlll and cloned into pETMAPF vector. The resultant clone was
screened with colony PCR and confirmed by restriction digestion. The clones were then
introduced into DE3 cells and were used for expression analysis using 1mM IPTG. The
induced cultures were pelleted, lysed by homogenizer and soluble and insoluble fraction was
separated by centrifugation. The samples were then analysed on SDS-PAGE. The fusion
expressed as a soluble protein was treated with enterokinase to get protein of interest.
In a further embodiment of the invention provided for a fusion protein DNA and the process
to expresses the proteins in soluble fraction when expressed as N terminal fusion protein.
The fusion DNA comprises of MAP protein with an enterokinase site (EK) at C terminus and
a 6X His tag at its N terminus. EK site ensures the generation of authentic N terminus of
protein of interest after cleavage of the fusion protein with enterokinase, while the N terminus
6X His tag aids in purification of the fusion protein through a single step of metal affinity
chromatography.
In an embodiment where several molecules ranging from small molecules like human IL-2
(14 kDa) to large molecules like PNGaseF (36 kDa) to a molecule having several disulphide
linkages like reteplase (9 disulphides) have been demonstrated to be successfully expressed
as MAP fusion proteins using the process of the invention. The proteins of the invention
fractionated into the soluble fraction of the E. coli cells. Thus providing for soluble fractions of
the protein of interest expressed by the process of the invention.
In another embodiment of the invention the protein of interest is obtained in correctly folded
form like PNGaseF protein was found to be active after enterokinase digestion.
The Enterokinase site may be optionally replaced by other cleavage enzymes like TEV
protease, Factor Xa and thrombin etc.
Other aspects and advantages of the present invention will be apparent upon consideration
of the following detailed description of preferred embodiments thereof.
Example 1: Cloning, expression and purification of MAP molecule
MAP molecule was amplified using following gene specific primers using E. coli cells
genomic DNA. The primers used were 5' CCG CCG GAA TTC CAT ATG GCT ATC TCA
ATC AAG ACC CCA GAA 3' and reverse primer which contains histidine 6x tag, 5' CCG

CCG GAA TTC AAG CTT TTA ATG ATG ATG ATG ATG ATG TTC GTC GTG CGA GAT
TAT CGC 3' and annealing temperature used was 50 °C for first five cycles and 60 °C for
remaining 25 cycles.. The amplified MAP gene was digested with Ndel and Hindlll and
cloned in pET21a vector.
Plasmid DNA was isolated from the cultures and restriction analysis was done to confirm the
release of insert by Nde1/Hindlll digestion. The resultant clones were designated as
pETMAP.
The clones were then introduced into DE3 cell line and were used for expression analysis.
The BL21(DE3) pETMAP clones were inoculated into 50 ml LB amp and kept in orbital
shaker at 37 °C. The cultures were induced with 1 mM IPTG and after 4 h were pelleted at
8000 rpm for 10 min. The pellets obtained were then lysed with homogenizer and soluble
and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The
samples were then analyzed on SDS- PAGE.
The MAP protein is expressed completely in soluble fraction (Fig 1). The MAP protein was
further purified using Nickel NTA agarose and the bound protein eluted from column was
pure (Fig 1) shows the purified MAP protein.
Example 2: Cloning and construction of MAP fusion vector
MAP gene was amplified using E. coli cells gene as template with following primers as
forward primer 5' CCG CCG GAA TTC CAT ATG GCT ATC TCA ATC AAG ACC CCA GAA
3' and reverse primer 5' CCG CCG GAA TTC AAG CTT TTA ATG ATG ATG ATG ATG ATG
TTC GTC GTG CGA GAT TAT CGC 3'. The amplified PCR product was cloned into pET21a
vector at Ndel BamHI sites. The clones were confirmed by restriction digestion. The
resultant vector was designated as pETMAPF and vector map is given in Fig 4.
The clones were introduced into BL21 A1 cell line and were inoculated in 50 ml LB amp and
induced after 1h with 13 mM arabinose and 1% lactose for 4h. The cell were pelleted and
disrupted in a cell disruptor. The lysed cell suspension was centrifuged at 13000 rpm for 20
min and the soluble and insoluble fractions were separated. The samples were analyzed on
a 13% SDS-PAGE.
It was found that MAP protein was completely restricted to the soluble fraction of the E. coli
cytoplasm.
This vector was designated as pMAPF and contains MCS site with BamHI, Hindlll, EcoRI,
Sacl, Sall, Xhol etc sites. This vector was used for cloning of different genes to get soluble
expressions for the respective proteins. The vector was designed in such a way that after
enterokinase digestion, the protein of interest will have authentic amino acid.
Example 3: Cloning and expression of reteplase molecule as MAP fusion

Reteplase (RTP) is a protein having high molecular weight 39 kDa containing 9 disulphide
bonds. RTP molecule was amplified from a synthetic template and digested with BamHI and
Hindlll and cloned into pETMAPF vector (MAP without stop codon). The primers used for
amplification were as follows forward primer 5' CCG CCG GGA TCC GAT GAT GAT GAT
AAA TCT TAC CAA GGC AAC AGC GAT TGC 3'and reverse primer 5' CCG GAA TTC AAG
CTT TTA CGG TCG CAT GTT GTC ACG AAT CCA 3' and annealing temperatures were 50
°C for first five cycles and 60 °C for remaining 25 cycles. So the resultant clone contains
MAPRTP fusion with enterokinase site before N terminus of RTP. The clone was confirmed
with restriction digestion.
The clones were then introduced into a BL21 (DE3) cell line and were used for expression
analysis. The BL21 (DE3) MAPRTP clone was inoculated into 250 ml LB medium with
ampicillin (amp) and kept in orbital shaker at 37 °C. The cultures were induced with 1 mM
IPTG and after 4 h were pelleted at 8000 rpm for 10 min. The pellets were suspended in 10
mM Tris CI obtained was then lysed with homogenizer and soluble and insoluble fractions
were separated by centrifuging at 13000 rpm for 20 min. The samples were then analyzed
on SDS- PAGE (Fig 2).
The MAP RTP fusion protein was expressed as a soluble protein. The RTP molecule was
expressed as a fusion of MAP protein was tested for activity using calorimetric assay and
blood clot lysis assay and was found to be active.
When MAPRTP fusion lysate was incubated with enterokinase, the fusion protein was
digested into two fragments MAP and RTP proteins (Fig 3).
Example 4: Cloning and expression of PNGase F molecule as MAP fusion
PNgase F gene (1065 bp) is 39 kDa protein is used in removal of carbohydrate moeitis from
glycoproteins. The PNGase F gene was subcloned into pETMAPF vector at EcoRI site from
a synthetic template. The resultant clone contains MAPPNGaseF fusion with enterokinase
site before N terminus. The clones were confirmed with restriction digestion.
The clones were introduced into DE3 cell line and were used form expression analysis. The
DE3 MAP PNGaseF clones was inoculated into 250 ml LB amp and after 2 h was induced
with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were pelleted
and lysed with homogenizer and soluble and insoluble fractions were separated by
centrifuging at 13000 rpm for 20 min. The MAP-PNGaseF fusion protein seen as 69 kDa
band was expressed in the soluble fraction as seen on SDS-PAGE gel (Fig 2).
The BL21 (DE3) MAP-PNGaseF lysate was incubated with enterokinase and both
MAP and PNGaseF proteins were observed in SDS-PAGE gel (Fig 3).
Example 5: Cloning and expression of IL-2 molecule as a MAP fusion

Interleukin 2 (IL-2) is a cytokine protein of 14 kDa size and used in therapeutic applications.
IL-2 gene was amplified from a synthetic template using forward primer 5' CCG CCG GGA
TCC GAT GAT GAT GAT AAA CCT ACT TCA AGT TCT ACA AAG 3' and 5' CCG GAA
TCC AAG CTT TCA AGT CAG TGT TGA GAT GAT GCT 3' digested with BamHI Hindi)! and
cloned into pETMAPF vector. The resultant clone contains MAPIL-2 fusion with enterokinase
site before N terminus. The clones were confirmed with restriction digestion.
The clones were introduced into DE3 cell line and were used form expression analysis. The
BL21(DE3) MAP IL-2 clones was inoculated into 250 ml LB amp and after 2 h was induced
with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were pelleted
and lysed with homogenizer and soluble and insoluble fractions were separated by
centrifuging at 13000 rpm for 20 min. The MAPIL-2 fusion protein was expressed in soluble
fraction as seen on SDS-PAGE gel (Fig 2).
Example 6: Cloning and expression of IFN molecule as MAP fusion
IFN gene was amplified from a synthetic template using forward primer 5' CCG CCG GGA
TCC GAT GAT GAT GAT AAA TGT GAC CTA CCA CAA ACC CAC 3' and reverse primer 5'
CCG CCG GAA TTC AAG CTT TTA TCA TTC CTT ACT TCT TAA ACT TTC 3'digested with
BamHI Hindlll and cloned into pETMAPF vector. The resultant clone contains MAPIFN
fusion with enterokinase site before N terminus. The clones were confirmed with restriction
digestion.
The clones were introduced into BL21 (DE3) cell line and were used form expression
analysis. The BL21(DE3) MAP IFN clones was inoculated into 250 ml LB amp and after 2 h
was induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells
were pelleted and lysed with homogenizer and soluble and insoluble fractions were
separated by centrifuging at 13000 rpm for 20 min. The MAPIFN fusion protein was
expressed in soluble fraction as seen on SDS-PAGE gel (Fig 2). The IFN protein was
released after enterokinase digestion of DE3 MAPIFN lysate (Fig 3). The most important
advantage with this vector is tRNA's for rare codons are not required since the expression of
MAPIFN fusion protein was achieved without supplementation of the specific rare codon
tRNA's.
Example 7: Cloning and expression of EGF as MAP fusion
Epidermal Growth factor (EGF) gene was amplified from a synthetic template using forward
primer 5' CCG CCG GGA TCC GAT GAT GAT GAT AAA AAT AGT GAC TCT GAA TGT
CCC CTG 3' and reverse primer 5' CCG CCG AAG CTT TAC GTA TTA GTG CAG TTC
CCA CCA CTT CAG 3' and digested with BamHI and Hindlll and cloned into pETMAPF

vector. The resultant clone contains MAPEGF fusion with enterokinase site before N
terminus. The clones were confirmed with restriction digestion.
The clones were introduced into BL21 (DE3) cell line and were used form expression
analysis. The DE3 MAPEGF clones was inoculated into 250 ml LB amp and after 2 h was
induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were
pelleted and lysed with homogenizer and soluble and insoluble fractions were separated by
centrifuging at 13000 rpm for 20 min. The MAP-EGF fusion protein was expressed in soluble
fraction (Fig 2)
Example 8: Cloning and expression of human enterokinase as MAP fusion
Human enterokinase gene was amplified from a synthetic template and digested with EcoRI
Hindlll and cloned into pETMAPF vector. The resultant clone contains MAPEK fusion with
enterokinase site before N terminus. The clones were confirmed with restriction digestion.
The clones were introduced into BL21 (DE3) cell line and were used form expression
analysis. The BL21(DE3) MAP EK clones was inoculated into 250 ml LB amp and after 2 h
was induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells
were pelleted and lysed with homogenizer and soluble and insoluble fractions were
separated by centrifuging at 13000 rpm for 20 min. The MAPEK fusion protein was
expressed in soluble fraction (Fig 2).
When the soluble MAP-EK fusion protein was purified with nickel column, the MAPEK fusion
protein was self cleaved into MAP protein and EK protein (Fig 3).
Example 9: Cloning and expression of IFN, IL-2, RTP and EK gene in pET21a vector
without fusion tags:
IFN, IL-2, RTP and EK gene were amplified and cloned at pET21a at Ndel Hindlll site to test
the expression of the proteins without any tags. RTP and EK proteins were expressed in BL
21 DE3 cell line were fractionated in insoluble fraction. IFN required rare codons to express
and were expressed only in insoluble fraction of BL21 DE3 codon plus cell line. Also, IL-2
was expressed at very low levels in insoluble fraction BL21 DE3 cell line (Fig 5).

We Claim:
1. A process for the preparation of heterologous protein in E. coli cells comprising the steps
of:
a) preparing a fusion DNA comprising a first DNA encoding MAP protein and a second DNA
fused in the frame encoding the heterologous protein of interest,
b) cloning of the vector comprising the fusion DNA of step a,
c) expressing the fusion protein in E. coli cells in soluble form,
d) obtaining protein from the fusion protein and
e) purifying the protein.
2. The process as claimed in claim 1, wherein the heterologous protein of interest is selected
from the group consisting of cytokines, growth stimulating factors, hormones, interferons,
interleukins and enzymes.
3. The process as claimed in claim 1, wherein the heterologous protein of interest is selected
from the group consisting of parathyroid hormone (1-34), parathyroid hormone (1-84),
reteplase, interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11 and GCSF.
4. The process as claimed in claim 1, wherein the fusion DNA further comprises an
Enterokinase cleavage site.
5. The process as claimed in claim 1, wherein the protein is purified using one or more
chromatographic purification technique selected from the group consisting of affinity
chromatography, metal affinity chromatography, hydrophobic interaction chromatography,
ion exchange chromatography and size exclusion chromatography.
6. A fusion DNA comprising a first DNA encoding MAP protein and a second DNA fused in
the frame encoding the heterologous protein of interest.
7. The fusion DNA as claimed in claim 6, having a nucleotide sequence of SEQ ID 1.
8. A fusion DNA comprising a fusion protein having the amino acid sequence of SEQ ID 2.

The present invention relates in general to a process of production of recombinant
heterologous proteins in E. coli expression system using E. coli, Yeast, fungi or mammalian
methionine amino peptidase (MAP) protein as a fusion tag. Further the invention relates to
the fusion proteins of MAP protein.