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1. WO2017130222 - PROCESS FOR EXTRACELLULAR SECRETION OF BRAZZEIN

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We claim,

1. A process for extracellular secretion of Brazzein having improved yield comprising:

(a) ligating a nucleotide sequence encoding a signal leader sequence conjugated to Brazzein with an expression vector to obtain a DNA construct and introducing the said construct into BL21(DE3) E. coli cells by transformation;

(b) culturing the transformed cells of step (a) carrying the recombinant construct in a culture medium in the presence of Dextrose and inducing protein expression with agents selected from IPTG or lactose for a period ranging from 2 hrs to 72 hrs to obtain secretion of Brazzein into the culture medium, and

(c) separating culture medium from cells 2-72 hours post induction and purifying Brazzein from the clarified medium..

2. The process according to claim 1, wherein Brazzein is selected from the group consisting of wild type III Brazzein and variants or multi-variants of the type III Brazzein.

3. The process according to claim 1, wherein the recombinant expression vector is selected from a pET vector or an expression vector having an IPTG inducible promoter.

4. The process according to claim 1, wherein the signal leader sequence is selected from the group consisting of pelB ompA, Bla, PhoA, PhoS, MalE, LivK, LivJ, MglB, AraF, AmpC, RbsB,MerP, CpdB, Lpp, LamB, OmpC, PhoE, OmpF, TolC, BtuB and LutA signal sequences.

The process according to claim 1, wherein the inducing agent is added culture medium in concentrations ranging from about 0.25mM to 5 mM.

6. The process according to claim 1, wherein culturing of cell is carried out at temperatures of between 25°C to 37°C.

7. The process according to claim 1, wherein the yield of Brazzein is ranging from about 0.5 g/1 to about 5g/l.

8. The process according to claim 1, where the expression host is selected from the group consisting of BL21(DE3) cell line and derivatives of the parental E.coli BL21 cell line.

9. The process according to claim 1, wherein the inducing agent is selected from the group consisting of Lactose and Lactose Analogues.

10. The process according to claim 1, where Dextrose is a component of the growth media prior to induction.