DESC:FIELD OF THE INVENTION
The present invention relates to a novel composition of culture media for bacteria. More specifically, the invention relates to a novel composition of chemically defined media for bacteria, more particularly, for Escherichia coli, for specifically increasing secretion of recombinant proteins/peptides.

BACKGROUND OF THE INVENTION
There are several target proteins having clinical or industrial applicability that are obtained using techniques, typically called as fermentation process. The fermentation process typically facilitates target protein synthesis in bacterial or in eukaryotic cell cultures in a controlled environment. However, once synthesized, there are often problems in recovering these recombinant proteins to reach substantial yields and its clinical or industrial applicability, in a useful form. For example, recombinant proteins expressed in bacteria often accumulate in the bacterial cytoplasm as insoluble aggregates known as inclusion bodies. Similarly, recombinant transmembrane proteins which contain both hydrophobic and hydrophilic regions are intractable to solubilization. Thus, there are several limiting factors that restrict the applicability of these target proteins in clinical or industrial application, because of lack of solubilization of these target proteins efficiently and in a cost effective manner.
One of the most important methods of obtaining recombinant proteins from the host cells is their secretion into the extracellular culture media. Culture media are used to grow cells in order to obtain desired components from them. However, there are several problems associated in this method. One of such problem is due to the fact that recombinant proteins are generally heterologous proteins (derived from organisms different from the host cells) and folding of these proteins in the hosts cells may not be appropriate for them to traverse the host membrane, thereby decreasing their secretion.
Another drawback associated, is the fact the incomplete purification of secreted target recombinant proteins, the impure target recombinant proteins has decreased availability of recombinant proteins due to undesired complexing with undefined peptides used in complex culture media used for growth of such host cells. Lysogenic Broth (LB) media is commonly used to grow Escherichia coli to promote growth and protein production. LB broth contains, per ml, 10 mg tryptone (a mixture of peptides formed by the digestion of casein with the pancreatic enzyme, trypsin), 5 mg yeast extract (an autolysate of yeast cells), and 5 or 10 mg NaCl. However, Lysogenic Broth is expensive and is comprised of non-defined animal, plant and microbial materials or extract. These non-defined materials or extracts vary from batch to batch and consistent bacterial growth in these media is difficult to achieve. The culture media used for growth of such host cells use materials derived from yeast, plants, or animal as sources Nitrogen, or Carbon in a culture media which makes it complex. There are a number of other difficulties associated with using such “complex” media, such as:
the exact compositions of the nitrogenous organic compounds varies between samples, and this inconsistency results in substantial variation in the batch-to-batch production level of the desired protein;
kinetic studies involving mass and energy balance play an important role in predictive mathematical model building of culture, which is necessary for scaling up the process, but the limited knowledge of the chemistry of the organic compounds in complex media prevents this;
residual proteinaceous materials in the final culture broth means that downstream processing (i.e. purification of the desired protein) is not always easy and culture proteins (e.g. serum proteins) may remain in the final product.
Moreover, pathogens such as prions and viruses have been identified as potential infectious agents that may reside in those animal or plant or microbial derived products. Many regulations now strongly address these concerns about using animal and plant derived or non-defined animal proteins in bacterial culture especially Escherichia coli cells.
To support the growth and multiplication of bacteria, a variety of components are essential to be included in the culture media. For example, glucose is basic energy sources that support bacterial growth and multiplication. Simply, breakdown of glucose provides resources for energy-generating pathways. The byproducts of these pathways are also the building blocks or sources for polypeptide synthesis. In addition, vitamins, amino acids and trace elements are also essential for robust cell. Trace elements are also important for the growth of animal cell and adding trace elements, such as Zinc, iron, selenium, copper, molybdenum, and manganese, etc., was important for cloning and continuous passage of bacterial cells in chemical defined media.
Hence, the present invention takes into account the drawbacks of the prior art and provides a novel composition of a chemically defined culture media for growth of bacteria which promotes secretion of recombinant proteins.

OBJECT OF THE INVENTION
The main object of the invention is to provide a novel composition of a chemically defined culture media for bacteria, more specifically E. coli.
Another object of the invention is to provide a novel composition of a chemically defined culture media for bacteria, more specifically E. coli, wherein secretion of recombinant proteins is enhanced.
Yet another object of the invention is to provide a novel composition of a chemically defined culture media for bacteria wherein the media is devoid of complex components to facilitate and increase the purification of secreted recombinant proteins.

SUMMARY OF THE INVENTION
The present invention provides a novel composition of chemically defined culture media for bacteria. More specifically, the invention relates to a culture media for enhancing secretion and facilitating purification of recombinant proteins and /or peptides expressed by host bacteria, more specifically, Escherichia coli.
The present invention relates a novel composition of chemically defined culture media for bacteria comprising of at least one carbon source, more specifically, glucose/dextrose; at one nitrogen source; at least one essential amino acid, more specifically, glycine; at least one positively charged amino acid, more specifically, arginine; thiamine; glycerol as a stabilizing agent; citric acid; at least one magnesium salt; at least one potassium salt; at least one source of phosphorus; at least one sodium salt; at least one calcium salt; salts of trace elements; at least one chelating agent, preferably EDTA; and at least one organic and inorganic acid or base for maintenance of pH of the media between 5.8-8.5, preferably Hydrochloric acid or Sodium hydroxide or Ammonium hydroxide to maintain pH of the media between 5.8-8.5.
The present invention relates a novel composition of chemically defined culture media for bacteria for enhancing secretion and facilitating purification of recombinant proteins expressed using an inducible bacterial expression vector, pBacSec-LC. pBacSec-LC is provided for expression and enhanced secretion of recombinant protein comprising of:
a secretory signal sequence which is a combination of a) at least one DNA sequence encoding a signal sequence of genes selected from the group consisting of pelB represented by Seq. ID 1 encoding amino acid sequence Seq. ID 9, ompA represented by Seq. ID 2 encoding amino acid sequence Seq. ID 11, yebF represented by Seq. ID 3 encoding amino acid sequence Seq. ID 10, and ompF represented by Seq. ID 4 encoding amino acid sequence Seq. ID 12; and b) at least one DNA sequence encoding a carrier peptide, preferably, DNA sequence encoding truncated yebF represented by Seq. ID 5 and Seq. ID 6.

BRIEF DESCRIPTION OF DRAWING
Figure 1a is a schematic showing the pBacSec-LC bacterial expression vector;
Figure 1b depicts a graphical representation of Gaussian Luciferase assay comparing secretion efficiency of E. coli cells grown in either the chemically defined media represented as M4 or the complex media LB and Terrific broth;
Figure 1c depicts a graphical representation of Gaussian Luciferase assay comparing secretion efficiency of E. coli cells grown in either M4 media or other chemically defined media represented as M1, M2, and M3 media;
Figure 1d depicts a representative image of SDS-PAGE with culture media of E. coli cells expressing 10kDa peptide encoded by recombinant vector containing a secretory signal peptide (ompF), an anchored protein, and a gene expressing 10kDa peptide;
Figure 2a depicts is graphical representation of growth curve of E. coli in M4 media in 3L fermenter stage;
Figure 2a depicts is graphical representation of rate of glucose consumption of E. coli in M4 media; and
Figure 3 depicts a representative image of SDS-PAGE with culture media of E. coli cells expressing 10kDa in different concentrations of NaCl in M4 media.

DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term “Bacteriophage” means a virus that infects and replicates within bacteria;
The term “EDTA” means the chelating agent Ethylenediaminetetraacetic acid; and
The term “LB” means Luria-Bertani medium, also known as Luria broth, Lennox broth or lysogeny broth.

The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the invention are indicated. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. The present invention is described fully herein with non-limiting embodiments and exemplary experimentation.
In main embodiment, the invention provides a novel composition of chemically defined media, for growth and culture of bacteria, more specifically, Escherichia coli, having a pH of 5.5-8.5. More particularly, the media provides a chemically defined media for efficient growth of bacterial cells and enhanced secretion of recombinant proteins from the host cells.
In another embodiment of the present invention, the media comprises of at least one carbon source, more specifically, glucose/dextrose. The carbon source is preferably in the range of 10-250 mM concentration. The media further comprises of glycerol as a stabilizing agent, wherein the ratio of glucose/dextrose and glycerol is between 1:0.25 to 1:1, preferably, 1:0.5. Presence of glycerol in media is to prevent the high shearing forces and protect bacteria from damage, and to reduce denaturation of recombinant protein.
The media further comprises of at least one nitrogen source, wherein the nitrogen source is an ammonium salt. The nitrogen source is preferably in the range of 5-50 mM concentration.
Further, the media comprises of citric acid in the range of 5 to 25 mM. Citric acid’s function in the said chemically defined media is to prevent infection of E. coli with bacteriophage. Infection of E. coli with bacteriophages is common problem in bacterial cultures which leads to decrease in number of live bacterial cells in the culture, this inversely affects the production of recombinant protein thereby reducing its production. Mechanistically, bacteriophages are very sensitive to citric acid and unable to infect the bacteria in the presence of Citric acid. The common LB broth used for recombinant protein production lacks citric acid and is not effective in preventing bacteriophage infection in E. coli.
The media further comprises of salts of magnesium, potassium, phosphorus, and sodium. The medium also comprises of salts of trace elements selected from the group to not limited to iron, cobalt, manganese, copper, boron, molybdenum, and zinc.
The media also comprises of at least one essential amino acid, more specifically, glycine which plays an important role in maintenance of membrane potential of bacterial cells and enhances protein secretion.
The media further comprises of at least one positively charged amino acid, more specifically, arginine which acts a chaperone molecule and enhances recombinant protein folding intracellularly, thereby reducing formation of inclusion bodies or aggregates and facilitates secretion of folded recombinant proteins extracellularly.
Glycine enhances the secretion, whereas Arginine aids in folding of protein before secretion. Together, Glycine and Arginine, help secretion of recombinant proteins.
Additionally, the media comprises of at least one chelating agent including but not limited to Ethylenediaminetetraacetic acid (EDTA), and at least one vitamin, more specifically, thiamine.
In another embodiment, the invention provides a chemically defined media for growth and culture of bacteria, wherein, the media is devoid of any complex undefined materials which tend to decrease the solubility of secreted recombinant proteins in the culture media and reduce the final amount of recombinant proteins purified.

EXAMPLE 1
COMPARISION OF COMPOSTIONS OF VARIOUS MEIDA FOR Escherichia coli GROWTH AND RECOMBINANT PROTEIN SECRETION
A. COMPOSITION OF MEDIA
Table 1 provides the novel composition of the chemical defined media (M4) in accordance with the present invention for growth and culturing of E. coli which is devoid of any complex undefined components, and enhances recombinant protein secretion and enables its purification.
Table 1 Composition of novel media M4 in accordance with present invention
Main Components of the media Concentration range M4 media composition (mM)
Citric Acid 5 to 25mM 8.84
KH2PO4 50 to 150mM 97.73
(NH4)2HPO4 10 to 50mM 30.2
NaCl 1 to 10mM 2.13
GLYCINE 1 to 10mM 1
GLYCEROL 10-100mM 54.29
ARGININE 0.5 to 10mM 1
CaCl2 0.01 to 1mM 0.09
MgSO4.7H20 1 to 10mM 5
DEXTROSE 20 to 200mM 69.5
KANAMYCIN 20 to 200mM 0.085
THIAMINE 0.001 to 1mM 0.06

Salts of Trace elements in media Gram per Litre Concentration (µM)
Fe(III) citrate 1 to 5g 40.86
CoCl2-6H2O 0.1 to 2g 1.92
MnCl2-4H2O 0.5 to 5g 7.57
CuCl2-2H2O 0.01 to 1g 0.87
H3BO3 0.1 to 1g 4.85
Na2MoO4-2H2O 0.01 to 1g 1.21
Zn acetate-2H2O 0.5 to 5g 7.08
EDTA 0.01 to 5g 2.87

The efficiency of M4 media in secretion of recombinant protein was compared to the regular LB broth and Terrific broth which are complex media. The table below, Table 2, provides the composition of LB and Terrific Broth.
Table 2: Compositions of complex media
LB broth Terrific broth
Tryptone 10 g/L Tryptone 12 g/L
Yeast extract 5 g/L Yeast extract 24 g/L
NaCl 5 or 10 g/L Glycerol 4ml/L
0.17 M KH2PO4 100 ml/L
0.72 M K2HPO4 100 ml/L

In order to test which components of the media affect the growth of E. coli and secretion of recombinant proteins, additionally 3 more chemically defined media were prepared, M1, M2, and M3; and each media was compared to the novel media M4. The comparative compositions of M1, M2, M3, and M4 media are provided in Table 3.
Table 3 Compositions of chemically defined media
Chemicals Concentration (mM)
Media -1 Media-2 Media-3 Media-4
KH2PO4 22
100 97.73
Na2HPO4 42 50
NH4CL 19
MGSO4 1 1
CaCl2 0.09 0.35 0.09 0.09
NaCl 9 80 2.13
Glycine 1
Arginine 1
(NH4)2SO4 15
K2HPO4 0.1
NH4Cl 20
Na2SO4 2.5
FeCl3 2 x 10-3
KCL 20
Tris 120
Thiamine 0.06 0.06 0.06 0.06
Glycerol (0.5%) 54.29
Citric acid 8.84
(NH4)2PO4 30.2
L-Tryptophan
L-Valine
L-Isoleucine
L-Leucine
Dextrose 69.5 69.5 69.5 69.5
MgSO4.7H20 5
Salts of Trace elements in media Concentration (µM)
Fe(III) citrate 40.86
CoCl2-6H2O 1.92
MnCl2-4H2O 7.57
CuCl2-2H2O 0.87
H3BO3 4.85
Na2MoO4-2H2O 1.21
Zn acetate-2H2O 7.08
EDTA 2.87

B. Expression of recombinant protein using pBacSec-LC expression vector
As depicted in Figure 1a, pBacSec-LC, is of around 6793 basepairs comprising of secretory signal sequence for efficient and enhanced secretion of recombinant protein which is in tandem with the secretory signal sequence.
The pBacSec-LC vector comprises of:
tac promoter and lac operator as inducible promoter;
an RBS;
a secretory signal sequence which is a combination of a) at least one DNA sequence encoding a signal sequence of genes selected from the group consisting of pelB represented by Seq. ID 1 encoding amino acid sequence Seq. ID 9, ompA represented by Seq. ID 2 encoding amino acid sequence Seq. ID 11, yebF represented by Seq. ID 3 encoding amino acid sequence Seq. ID 10, and ompF represented by Seq. ID 4 encoding amino acid sequence Seq. ID 12; and b) at least one DNA sequence encoding a carrier peptide, preferably, DNA sequence encoding truncated yebF represented by Seq. ID 5 and Seq. ID 6;
DNA sequence encoding 6-His tag and FLAG tag which are affinity tags;
DNA sequence encoding recombinant protein;
a gene terminator for transcriptional termination of recombinant protein;
an ori sequence to enable replication of vector in E. coli;
a lac operon under an inducible promoter as a selectable marker for blue-white recombinant colony selection, and to make the vector inducible; and
a kanamycin resistance gene as an antibiotic selectable marker.
Table 4 provides the DNA sequence encoding the signal sequence or the carrier peptides of pBacSec-LC vector
SEQ. ID No. DNA sequence

1 ATGAAATACCTGTTACCTACCGCGGCTGCGGGGCTGCTGCTGTTAGCAGCTCAGCCGGCAATGGCT
2 ATGAAGAAGACCGCGATTGCGATTGCGGTGGCGCTGGCGGGTTTTGCGACCGTGGCGCAGGCG
3 ATGAAAAAGCGTGGTGCGTTCCTGGGCCTGCTGCTGGTTAGCGCGTGCGCGAGCGTGTTTGCG
4 ATGATGAAGCGCAATATTCTGGCAGTGATCGTCCCTGCTCTGTTAGTAGCAGGTACTGCAAACGCT
5 GCGAACAACGAAACCAGCAAGAGCGTGACCTTTCCGAAATGCGAAGATCTGGATGCGGCGGGTATTGCGGCGAGCGTTAAGCGTGACTACCAGCAAAAC
6 GCGAATAATGAGACCAGCAAAAGCGTGACCTTTCCGAAGGCGGAGGACCTGGATGCGGCGGGTATTGCGGCGAGCGTTAAACGTGACTACCAGCAAAAC
Peptide Sequence
7 ANNETSKSVTFPKCEDLDAAGIAASVKRDYQQN
8 ANNETSKSVTFPKAEDLDAAGIAASVKRDYQQN
9 MKYLLPTAAAGLLLLAAQPAMA
10 MKKRGAFLGLLLVSACASVFA
11 MKKTAIAIAVALAGFATVAQA
12 MMKRNILAVIVPALLVAGTANA

C. EFFECT OF MEDIA ON SECRETORY EFFICIENCY OF E. coli
E. coli strains, NEB 5-alpha and BL21(DE3) were used for experimentation and luciferase assay.
E. coli was transformed with recombinant bacterial expression vector, pBacSec-LC vector comprising of secretory signal sequence in the combination of Seq. ID 4 and Seq. ID 6 operably linked toDNA sequence of Guassia luciferase for conducting the luciferase assay. After transformation the cells were grown in either media M1, M2, M3, or M4 chemically defined media or LB broth or terrific broth under shaking conditions and tested for efficiency of secretion of Guassia luciferase using an assay kit.
Guassia luciferase assays were performed using Pierce Gaussian Luciferase glow assay kit. Media was collected from culture after 5 hours of induction using Isopropyl ß- D -1-thiogalactopyranoside (IPTG) and luciferase activity measured from media as described in manufacturer’s protocol.
As depicted in Figure 1b, M4 media shown maximum efficiency in secretion of recombinant Guassia luciferase compared other complex medial or Terrific. Figure 1c, further provides that M4 media composition is most efficient among the chemically defined media tested.
E. coli was also transformed with IPTG inducible recombinant vector containing a secretory signal peptide (ompF), an anchored protein, and a gene expressing 10kDa peptide. Samples were also collected 5 hours post induction (IPTG) i.e 300µL of culture, centrifuged at 14K rpm for 3 minutes to separate cells and culture media to check expression and secretion of protein. The media was further analyzed using SDS-PAGE. As depicted in Figure 1d, recombinant peptide of 10kDa was secreted by E. coli grown in M4 media compared to others.
These results clearly showed enhanced efficiency of M4 media on secretion of recombinant peptide.
Growth of E. coli was also measured in M4 media in 3L fermenter stage. As depicted in Figure 2a, the growth curve is a normal curve with increasing cells number over period of time.
Growth was also measured using rate of glucose consumption using Glucometer. As depicted in Figure 2b, amount of glucose in media reduced over period of time, suggesting consumption.
These experiments proved proof for medium scale fermentation process and suggested that the same conditions can be scaled up for large-scale fermentation.

EXAMPLE 2
EFFECT OF AMINO ACIDS IN MEDIA M4
Several amino acids in the media M4 were tested for their efficiency in enhancing protein secretion from E coli. Amino acids such as 0.01 to 0.1% L-Tryptophan, 0.01 to 0.1% L-Valine, 0.002 to 0.02% L-Isoleucine and 0.002 to 0.02% L-Leucine, were also tested. However, only L-arginine in the range of 0.5 to 10mM, and Glycine in the range of 1 to 10mM gave good growth and higher yield recombinant proteins.
EXAMPLE 3
EFFECT OF CONCENTRATION OF NaCl IN M4 MEDIA EFFICIENCY OF SECRETION OF RECOMBINANT PROTEINS
NaCl concentration was varied in M4 media and efficiency of secretion of 10kDa peptide was tested by SDS-PAGE as explained above.
As depicted in Figure 3, concentrations equivalent to 2.5 mM works best for secretion of recombinant peptide. Concentration of 5 mM and 10 mM reduce the secretory efficiency.
LB broth (Lennox) have 85 mM NaCl, whereas LB broth (Miller) has 171 mM NaCl. Terrific Broth composition does not have any NaCl. The above results clearly suggest that 2.5 mM of NaCl in media composition works best for enhancing secretion of recombinant protein which is provided by M4 chemically defined media and not by the regularly used complex media LB or Terrific.
While certain exemplary embodiments have been described and shown in the accompanying drawings, it is to be understood that such embodiments are merely illustrative of, and not restrictive on, the broad invention, and that this invention not be limited to the specific constructions and arrangements shown and described, since various other changes, combinations, omissions, modifications and substitutions, in addition to those set forth in the above paragraphs, are possible. Those skilled in the art will appreciate that various adaptations and modifications of the just described embodiments can be configured without departing from the scope and spirit of the invention.
,CLAIMS:CLAIMS
We claim,
1) A composition of chemically defined culture media having pH in the range 5.8-8.5 for growth of bacterial cells and for enhancing secretion of recombinant protein by bacteria, more specifically, E. coli, comprising of:
at least one carbon source at a concentration around 30-300mM, more specifically, glucose/dextrose;
at one nitrogen source at a concentration around 10-50mM;
glycerol as stabilizing agent at a concentration around 10-100 mM;
citric acid at a concentration around 5-25mM;
at least one essential amino acid at a concentration around 1-10mM;
at least one positively charged amino acid at a concentration around 0.5-10mM;
thiamine at a concentration around 0.001-1mM;
at least one organic and inorganic acid or base for maintenance of pH;
at least one magnesium salt at a concentration around 1-10mM;
at least one potassium salt at a concentration around 50-150mM;
at least one source of phosphorus at a concentration around 50-150mM;
at least one sodium salt at a concentration around 1-10mM;
at least one calcium salt at a concentration around 0.01-1mM;
salts of trace elements; and
at least one chelating agent at a concentration around 0.01-5 g/L, more specifically, Ethylenediaminetetraacetic acid (EDTA)
wherein,
the composition of the media is as defined for M4 media;
the combination of glucose/dextrose and glycerol is in the ratio of 1:0.25 to 1:1, preferably, 1:0.5;
the essential amino acid is glycine;
the positively charges amino acid is arginine; and
the sodium salt is NaCl in the range of 1-10mM, more preferably, 2.13mM.
2) The chemically defined culture media as claimed in claim 1, wherein, the medium comprises of salts of trace elements selected from the group to iron, cobalt, manganese, copper, boron, molybdenum, and zinc.
3) The chemically defined culture media as claimed in claim 1, wherein,
media enhances recombinant protein secretion by bacteria expressed using an inducible bacterial expression vector, pBacSec-LC, comprising of a secretory signal sequence which is a combination of a) at least one DNA sequence encoding a signal sequence of genes selected from the group consisting of pelB represented by Seq. ID 1 encoding amino acid sequence Seq. ID 9, ompA represented by Seq. ID 2 encoding amino acid sequence Seq. ID 11, yebF represented by Seq. ID 3 encoding amino acid sequence Seq. ID 10, and ompF represented by Seq. ID 4 encoding amino acid sequence Seq. ID 12; and b) at least one DNA sequence encoding a carrier peptide, preferably, DNA sequence encoding truncated yebF represented by Seq. ID 5 and Seq. ID 6;
the DNA sequence encoding the recombinant protein is operably linked to the secretory signal sequence; and
pBacSec-LC vector comprises of lactose or lactose analogues including IPTG inducible lac operon.
4) The chemically defined culture media as claimed in claim 1, wherein, the media enhances recombinant protein secretion compared to complex media and other chemically defined media.
5) The chemically defined culture media as claimed in claim 1, wherein, the media enhances secretion of recombinant protein by bacteria, more specifically E. coli, and facilitates purification of recombinant protein.