DESC:FIELD OF THE INVENTION
The present invention relates to a method and system for enhanced production, secretion and separation of recombinant polypeptides from bacteria, more specifically E. coli. More specifically, the method and system provide a separation system and method for alternating tangential filtration flow for continues removal of secreted recombinant protein from a culture media.

BACKGROUND OF THE INVENTION
Conventionally, most pharmaceutical fermentation products have been derived from bacteria and fungi grown in stirred tank fermenters ranging in tens of thousands of liters. Moreover, with the advent of recombinant DNA techniques in the mid-1970s and the emergence of the biotechnology industry in the late 1970s, it was assumed that production of the new class of pharmaceuticals would be done in the same fermenters.
However, the major limitation of use of bacteria for production of such industrially important molecules is lack of post-translational modifications of these molecules lacking which rendered the molecules inactive. Hence, a lot of research focused on use of mammalian cells for production of the same and several fermentation processes were developed. Moreover, use of bacterial cells for production of recombinant proteins also had other limitations such as formation of intracellular aggregates or inclusion bodies, difficulty in extraction of intracellular produced recombinant proteins etc. The recombinant proteins that were secreted which were subjected to deficiencies in down-stream processing for purification of the same due to use of complex media. Hence, although bacterial systems are easier to work with as compared to mammalian/plant cell systems, they have not been used widely for producing mammalian proteins/peptides owing to the several limitations cited above.
It is typically a batch fermentation method used for bacterial systems to obtain useful recombinant proteins. For every cycle repetition for next batch is required, the method requires compete removal of the fermentation broth, the need for repeated discharge, tank washing, sterilization and other materials which is tedious, time consuming and expensive. There is a short production time and manpower, material and power consumption of large defects.
Recently, there have been developments in generation of expression vectors specifically designed to increase secretion of recombinant proteins in properly folded forms,. Similarly, developments have been made to provide media which enhances secretion of recombinant proteins and eases the purification of the same. Further systems have been developed to maximize the output for industrial scale production of secretory recombinant proteins/peptides, thereby developing an efficient fermentation processes, as described herein.
WO2005095578A1 discloses a continuous perfusion and alternating tangential flow cell culturing process specifically suited for culturing cells which tend to aggregate. The invention describes culturing of a cell culture comprising cell culture medium and cells, wherein cell culture medium is added to the cell culture, wherein the cell culture is circulated over a filter module comprising hollow fibers resulting in an outflow of liquid having a lower cell density than the cell culture and wherein the flow within the filter module is an alternating tangential flow. It has surprisingly been found that by perfusion culturing of animal, in particular mammalian, cells or yeast cells according to the invention, extremely high viable cell densities can be obtained, whereas the cell culture further displays an extremely high cell viability.
EP1498475A1 discloses a continuous plant cell bioreactor comprising a fermentation chamber, an oxygenating inlet enabling oxygenating fluid to enter the chamber, a cell media inlet enabling cell media to enter the chamber, and a plant cell supernatant outlet enabling cell supernatant to exit the chamber, wherein the cell media inlet and the supernatant outlet are located substantially one opposite the other. The method and apparatus can be used to continuously produce recombinant therapeutic proteins from plant cell culture. The suspension of transgenic plant cells are suitable for expressing a recombinant polypeptide or peptide fragment, preferably selected from N-glycosylated and N-unglycosylated polypeptides or peptide fragments.
CN102382794A discloses a method of operating a perfusion cultured mammalian cell, comprising the steps of: (1) The target cell line in the initial stage of the culture medium, progressively larger, and (2) cell culture in a fermenter stage, which characterized in that the primary culture cells were cultured to the logarithmic phase of the cell growth phase temperature of 36-37 ° C, pH is 7. 1-72, stirring speed 150rpm; when the cell density reached 8X 106ceIls/mL, the temperature is adjusted 31-33 ° C, adjusted to pH 6. 9-7. 0, adjust the stirring speed 80_100rpm, wherein said target cell line selected from CHO cells, NSO cells, VERO cells, or BHK cells and the like commonly used mammalian host cell.
The prior art disclosed here above do not provide application of alternating tangential filtration flow for continues removal of secreted recombinant protein from a culturing suspension for recombinant polypeptides in bacteria with customized separation system for maximizing the output of the fermentation systems.

OBJECT(S) OF THE INVENTION
Accordingly, the present invention provides a technical advantage over the inventions present in the state of the art.
The main object of the present invention is to provide a system and method for enhanced production, and secretion of recombinant polypeptides in bacteria, more specifically E. coli, with a customized perfusion-based fermentation and separation system for alternating tangential filtration flow for continues removal of secreted recombinant protein from a culturing suspension; wherein, the method comprises the steps of expressing the recombinant protein using vector pBacSec-LC, and carrying out the fermentation process using a chemically defined media and employing a perfusion-based fermentation and separation system to filter the recombinant proteins.
Yet another object of the present invention is to provide the separation system comprising a filter module with hollow fibers, selected from materials such as polysulphone, Methyl Ester or Cellulose ester having a porosity of between 0.4 µm and 0.1 µm, e.g. a porosity of 0.2 µm.
Yet another object of the present invention is to provide the filter module comprising membranes wherein the membranes have a molecular weight cut-off pore size of between 500,000 and 5,000 Da, between 20,000 and 1,000 Da or below 5,000 Da.

SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method and system for enhanced production and secretion of recombinant polypeptides in bacteria, more specifically E. coli. The method and system also provide a separation system for alternating tangential filtration flow for continues removal of secreted recombinant protein from a culturing suspension in the fermenter.
In an embodiment of the present invention, the invention provides a method for culturing a bacterial strain, more specifically E. coli, in a perfusion-based fermentation and separation system comprising the steps of: a) adding a liquid growth medium to a fermenter; b) seeding the growth medium with recombinant bacteria transformed with recombinant vector carrying the gene encoding recombinant protein to form a culture medium; c) growing the bacteria in perfusion suspension culture, wherein the culture medium including the bacteria is circulated over a separation system in alternating tangential flow, and the separation system removes a filtrate containing spent medium containing heterologous recombinant protein from the culture medium and retains the bacteria in the culture medium for continuous production; the separation system comprises hollow fibers made of polysulphone and Methyl Ester or Cellulose ester having a porosity between 0.4 µm and 0.1 µm, more particularly a porosity of 0.2 µm; and the membranes have a cut-off pore size of between 500,000 and 5,000 Da, between 20,000 and 1,000 Da or below 5,000 Da.
In another embodiment of the present invention, the system provides a feed tank; a feed pump; a level sensor, a motor; a fermenter vessel; a magnetic levitation pump, separation module, harvest pump, and a harvest tank, wherein the separation module further comprises of hollow fiber membranes suitable for the removal of secreted recombinant protein from the culture medium. The separation module comprises of a filter module comprising hollow fibers, made of polysulphone, Methyl Ester or Cellulose ester having a porosity of between 0.4 µm and 0.1 µm, e.g. a porosity of 0.2 µm.
In yet another embodiment, the separation system comprises a filter module comprising membranes wherein the membranes have a molecular weight cut-off pore size of between 500,000 and 5,000 Da, between 20,000 and 1,000 Da or below 5,000 Da.
In yet another embodiment a separation system, comprising a feed tank (101); a feed pump (102); a level sensor (103); a motor (104); a fermenter vessel (105); a magnetic levitation pump (106); separation module (107); harvest pump (108), and a harvest tank (109), wherein, the separation module (107) comprises a filter module (110) of hollow fiber membranes (111) suitable for the removal of secreted recombinant protein from the culture medium; the filter module (110) of hollow fiber membranes (111) comprises of polysulphone and Methyl Ester or Cellulose ester having a porosity between 0.4 µm and 0.1 µm, more particularly a porosity of 0.2 µm; and the membranes have a cut-off pore size of between 500,000 and 5,000 Da, between 20,000 and 1,000 Da or below 5,000 Da.
In yet another embodiment, a recombinant vector, 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.
In yet another embodiment, the invention provides the composition of a chemically defined media for initial batch preparation of recombinant bacteria expressing recombinant protein and a perfusion media for growth and secretion of recombinant protein during continuous fermentation process.
BRIEF DESCRIPTION OF THE FIGURE(S)
A complete understanding of the system and method of the present invention is obtained by reference to the following figure(s):
Figure 1 elucidates the schematic diagram of the system;
Figure 2 elucidates the growth curve of the bacterial cells fermented in the system after perfusion;
Figure 3 is a schematic of the recombinant vector pBacSec-LC for enhanced secretion of recombinant protein;
Figure 4 is a representative image of SDS-PAGE analysis of 10kDa recombinant protein secretion in fermenter using pBasSec-LC vector in E. coli after 4 hours of induction with 0.25mM IPTG in Batch method and after 7 hours in perfusion method; and
Figure 5 is a representative image of SDS-PAGE analysis of 10kDa recombinant protein secretion in fermenter using pBasSec-LC vector in E. coli after different time points of induction in perfusion method.

DETAILED DESCRIPTION OF THE INVENTION

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.
Figure 1 elucidates the schematic diagram of perfusion-based fermentation and separation system, the system comprising a feed tank (101); a feed pump (102); a level sensor (103); a motor (104); a fermenter vessel (105); a magnetic levitation pump (106); separation module (107); harvest pump (108), and a harvest tank (109), wherein hollow fiber column in the separation module (107) comprises a filter module (110) of hollow fiber membranes (111) suitable for the removal of secreted recombinant protein from the culture medium.
The feed tank (101) stores media that are pumped via a feed pump (102) to a fermenter vessel (105). The fermenter vessel (105) holding the culturing suspension comprising of inoculum and the media is agitated by a motor (104). The inoculum is preferentially bacteria. The fermenter vessel (105) is continuously or intermittently drained via a magnetic levitation pump (106). The level of media is maintained via a level sensor (103). The drained liquid is further passed through a filter module comprising hollow fiber membranes (111). The culturing suspension in the fermenter vessel (105) is circulated over a separation system in alternating tangential filtration flow. The separation module (107) removes secreted recombinant protein from culture media continuously. The suspension filtered via a filter module (110) comprising hollow fiber membranes (111) suitably removes the recombinant protein from the culture medium and inoculum. The filtered inoculum is re-introduced into the fermenter vessel and the liquid media is pumped via a harvest pump (108) to the harvest tank (109).
In an embodiment of the present invention the separation system comprises a filter module comprising hollow fibers of polysulphone, Methyl Ester or Cellulose ester having a porosity of between 0.4 µm and 0.1 µm, with porosity of 0.2 µm.
In yet another embodiment, the separation system comprises a filter module comprising membranes wherein the membranes have a molecular weight cut-off pore size of between 500,000 and 5,000 Da, between 20,000 and 1,000 Da or below 5,000 Da.
Figure 2 elucidates the growth curve of the E. coli fermented in the system after perfusion. After first hour of induction, perfusion was started to supplement limiting nutrients in order to keep the cell proliferation at minimal rate and dedicate most of the time for recombinant protein production.
In an embodiment of the present invention the method provides method for culturing a bacterial strain, E. coli in a fermenter comprising the steps of: a) adding a liquid growth medium to a fermenter; b) seeding the growth medium with the bacteria to form a culture medium; c) growing the bacteria in perfusion suspension culture, wherein the culture medium including the bacteria is circulated over a separation system in alternating tangential flow, and the separation system removes a filtrate containing spent medium containing heterologous recombinant protein from the culture medium and retains the bacteria in the culture medium for continuous production.
Further, in another embodiment of the present invention a recombinant vector, pBacSec-LC, is provided for expression of recombinant protein. As depicted in Figure 3, 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 1 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

Still further, the invention provides a chemically defined media for growth and fermentation of bacterial cells for secretion and separation of recombinant protein. The media comprises 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;
glycine at a concentration around 1-10mM;
arginine 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).
Table 2: Composition of chemically defined media for growth and fermentation of recombinant bacteria for secretion and separation of recombinant protein
Main Components of the media Concentration range
Citric Acid 5 to 25mM
KH2PO4 50 to 150mM
(NH4)2HPO4 10 to 50mM
NaCl 1 to 10mM
GLYCINE 1 to 10mM
GLYCEROL 10-100mM
ARGININE 0.5 to 10mM
CaCl2 0.01 to 1mM
MgSO4.7H20 1 to 10mM
DEXTROSE 20 to 200mM
KANAMYCIN 20 to 200mM
THIAMINE 0.001 to 1mM

Salts of Trace elements in media Gram per Litre
Fe(III) citrate 1 to 5g
CoCl2-6H2O 0.1 to 2g
MnCl2-4H2O 0.5 to 5g
CuCl2-2H2O 0.01 to 1g
H3BO3 0.1 to 1g
Na2MoO4-2H2O 0.01 to 1g
Zn acetate-2H2O 0.5 to 5g
EDTA 0.01 to 5g

EXAMPLE 1
PERFUSION BATCH PROTOCOL
Transformed cells are prepared using E. coli BL21(DE3) taking appropriate plasmid by giving heat shock and spreading them on selective antibiotic LB agar plate followed by incubation for 12h in incubator at 37°C. Growth media, as provided in table 3, is prepared by taking media components and dissolving in deionized water and adjusted to pH 6.90 using 5N NaOH. Growth Media is sterilized at 121°C with 45 minutes holding time in autoclave. All the other media component that are heat liable are filter sterilized i.e. Thiamine, Kanamycin, Trace elements. Dextrose and MgS04.7H2O are separately autoclaved. Pre-culture is prepared by inoculation of colony from LB agar plate into shake flask. Filter sterilized media components i.e. Thiamine, Kanamycin 50mg/Lit, Trace elements 1000x were added along with 1% Dextrose, MgSO4.7H20- 5mM, yeast extract 0.2%. Incubated the flask in shaker incubator at 37°C at 225 rpm for overnight. Bioreactor vessel packed with pre-calibrated pH probe, antifoam sensor along with new membrane fitted to dissolved oxygen (DO) probe after refilling with Oxylyte, sparger pipe and exhaust pipe fitted with 0.2µ hydrophobic vent filter each, baffle. Growth media is poured into vessel excluding volume corresponding to reconstitutable media components. Vessel is autoclaved at 121°C, 45 min holding time, 15 psi, later cooled to room temperature and attached to Control unit. Vessel is purged with 0.1 SLPM of air overnight for DO probe polarization. DO is calibrated just before addition of pre-culture. Reconstitution of media is done before addition of pre-culture. Dosing bottle are attached, and tubing’s are primed. All the parameters are set in Control unit i.e. pH set to 6.90 with 0.1% Dead band, DO set to 30% in cascade mode, Acid/Base in auto mode, antifoam sensor with 0.1% Sigma Antifoam 204, Temperature set to 37°C. DO is controlled in cascade mode with Agitation, Gas flow per minute, % of pure oxygen purging. Initial RPM is set to 200. Pre-culture is aseptically transferred into bioreactor. First hour pH is maintained by attaching dosing bottle with 3N NaOH Rest of the batch pH is maintained by 4M Liquid Ammonia. MgSO4.7H20 is reconstituted aseptically for three doses from zeroth hour of fermentation. Hourly sampling is done to check cell density by UV/Vis Spectrophotometer and residual glucose by glucometer. After 5 hours or batch with residual glucose of ~5 g/L is picked as ideal time point to induce the culture with 0.2mM IPTG. Sterile 500kD Hollow fiber is connected to perfusion equipment and equilibrated with Sterile growth media, later tubing’s are connected to bioreactor. 30 minutes after post induction perfusion is started, here culture is drawn out of vessel automatically by pump passed through hollow fiber, retentate from the hollow fiber is sent back to bioreactor. Harvest is collected from permeate end of hollow fiber by setting pump speed to ~ 16.6 ml/minutes and simultaneously at same flow rate perfusion media is added into vessel. Total fermentation is run till the completion of perfusion media. After completion of feeding perfusion media, culture is concentrated to 2/3rd of batch volume by drawing 1/3rd of permeate and batch is terminated. Post induction hourly samples are drawn to check expression and secretion of recombinant protein. Hollow fibre is sanitized by 0.5N NaOH and stored in 0.1N NaOH.
Table 3: COMPOSITION OF THE MEDIA
Initial Batch Media Perfusion Media
S.NO Components Molarity (mM) Molarity (mM)
1 Citric Acid 8.84 8.84
2 KH2PO4 97.73 97.73
3 (NH4)2HPO4 30.2 30.2
4 NACL 2.13 2.13
5 GLYCINE 1 1
6 GLYCEROL 54.29 54.29
7 ARGININE 1 1
8 CACL2 0.09 0.09
9 MGSO4.7H20 5 1
10 DEXTROSE 139 13.9
11 KANAMYCIN 0.05 0.05
12 THIAMINE 0.06 0.06
13 IPTG 0 0.2

EXAMPLE 2
EFFICICENY OF SECRETION OF RECOMBINANT PROTEIN USING PERFUSION METHOD
Efficiency of recombinant protein secretion (10kDa protein) using perfusion method was analyzed. Recombinant E. coli transformed with pBacSec-LC vector with secretory signal combination of Seq. ID 4 and Seq. ID 6 was either grown under batch method or perfusion method.
Perfusion method of fermentation is explained in Example 1.
Whereas, batch method of fermentation involves,
i. Preparation of starter culture: 3ml of autoclaved growth media having pH of 6.90 is taken in sterile snap cap tube. Single CFU picked from Luria-Bertani agar plate and inoculated aseptically into growth media and incubated overnight at 37°C with 225 rpm in rotatory incubator.
ii. Shake flask culturing: Overnight starter culture is inoculated into 3L growth media (1:25 dilutions) having pH of 6.90. Flasks were incubated in rotatory shaker incubator maintained at 37°C, 225 rpm. After 4 hours of incubation OD600 reached ~1.8 to 2.0. Cells were induced with 0.2mM Isopropyl ß-d-1-thiogalactopyranoside (IPTG), and inducer of lac operon. The media samples were collected by separating cells and media.
Media samples were collected and analyzed after 4 hours of induction with 0.25mM IPTG in Batch method and after 7 hours in perfusion method. In batch method 3L media of collected whereas in perfusion 10 L media was collected for comparison. The media samples were compared by SDS-PAGE analysis for secreted recombinant protein.
As depicted in Figure 4, the recombinant protein concentration from 3L media of batch method fermentation and 10L media from perfusion-based method fermentation is almost similar. This strongly suggests that the perfusion method is as efficient as batch method. However, batch methods are difficult to scale up and have several other limitations such a nutrient depletion, toxic waste accumulation etc., whereas a perfusion-based fermentation method is highly efficient in scaling up as the yield is not affected. This was further confirmed by yield of recombinant protein in perfusion method after different time point. As depicted in Figure 5, recombinant protein was consistently produced and secreted as the time increased in perfusion method and it also was higher even after 12 hours of perfusion method fermentation. This suggested that perfusion method is suitable for long time production and secretion of recombinant protein.
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 perfusion-based fermentation system for enhanced continuous production, secretion and separation of recombinant polypeptides from bacteria, more specifically E. coli, in a culturing media comprising a feed tank (101); a feed pump (102); a level sensor (103); a motor (104); a fermenter vessel (105); a magnetic levitation pump (106); separation module (107); harvest pump (108), and a harvest tank (109), wherein, the separation module (107) comprises a filter module (110) of hollow fiber membranes (111) suitable for the removal of secreted recombinant protein from the culture medium; and the filter module (110) of hollow fiber membranes (111) comprises of polysulphone and Methyl Ester or Cellulose ester having a porosity between 0.4 µm and 0.1 µm, more particularly a porosity of 0.2 µm; and the membranes have a cut-off pore size of between 500,000 and 5,000 Da, between 20,000 and 1,000 Da or below 5,000 Da.
2. The perfusion-based fermentation system as claimed in claim 1, wherein, the culture medium including the bacteria is circulated over a separation system in alternating tangential flow, and the separation system removes a filtrate containing spent medium containing recombinant protein from the culture medium and retains the bacteria in the culture medium for continuous production.
3. A method for enhanced continuous production, secretion and separation of recombinant polypeptides from bacteria, more specifically E. coli, the comprising of the steps:
a) expression of recombinant polypeptide using a bacterial expression vector, pBacSec-LC; and
b) production of the recombinant polypeptide using a continuous fermentation, and separation system in a chemically defined media,
wherein
the 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 fermentation process is carried out using chemically defined culture media having pH in the range 5.8-8.5 for growth of bacterial cells and for enhancing recombinant protein secretion by bacteria 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;
glycine at a concentration around 1-10mM;
arginine 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);

the separation module (107) comprises a filter module (110) of hollow fiber membranes (111) suitable for the removal of secreted recombinant protein from the culture medium; and

the filter module (110) of hollow fiber membranes (111) comprises of polysulphone and Methyl Ester or Cellulose ester having a porosity between 0.4 µm and 0.1 µm, more particularly a porosity of 0.2 µm; and the membranes have a cut-off pore size of between 500,000 and 5,000 Da, between 20,000 and 1,000 Da or below 5,000 Da.