Extraction and purification of product from fermentation is known as Downstream Processing ( DSP) or Product Recovery
It is an essential step in the manufacture of pharmaceuticals product
Cost of the product is determined by the DSP involved
2. Introduction
2
Extraction and purification of product from
fermentation is known as Downstream
Processing ( DSP) or Product Recovery
It is an essential step in the manufacture of
pharmaceuticals product
Cost of the product is determined by the DSP
involved
3. [
3 Source - Glick
Upstrea
m
process
Downstream
process
7. Types of Filtration
7
Based on the particle sizes to be separated
Source – Nandakisor et.al.
8. Centrifugation
8
Principle - density differences between the
particles to be separated
Separating solid particles from liquid phase
Source – Nandakisor et.al.
11. Disadvantages
11
Whole cell contents are released out which
makes it difficult to separate out product of
interest from the mixture.
Cell lysis increases viscosity of the solution
making it difficult to process in the further steps.
Product released into harsh environment causing
the product to lose stability or activity.
Enzymatic cell-disruption in large scale can be
expensive
12. 3. Concentration
12
The commonly used techniques for concentrating
biological products are,
Evaporation
Liquid-liquid extraction
Membrane filtration
Precipitation
Adsorption
15. 5.Formulation
15
Maintenance of activity and stability of a
biotechnological products during storage and
distribution
Stabilizing additives - prolong the shelf life of
protein. (stabilizers include sugars , salts,
polymers and polyhydric alcohols)
Proteins may be formulated in the form of
solutions, suspensions or dry powders
17. Problems in DSP of rDNA
product
17
S.N
o
Problems Solutions
1 Protein stability - depends
on the susceptibility of the
protein for proteolytic
decomposition
•lon-minus mutants from E.Coli k12 strains
•Synthesis of fusion protein
2
Recombinant proteins are
often found as insoluble
aggregates in the
cytoplasm. These
accumulations of solid
insoluble proteins are
called inclusion bodies
•Force protein secretion
•Changing the specific properties of the target
protein
•Distribution of the charge
•Fusion of heterologous gene with soluble
protein
•Fusion of heterologous gene with chaperon
3
Separation of Cells and
Cell Disruption – loss of
the target protein
•kil-gene of the plasmid ColE1 may yield
complete lyses of the cells. kil-gene, under the
control of the lac-promoter may lyse the cells
18. Contd.
18
S.N
o
Problems Solutions
4 Localization – recombinant
protein may be lethal to the
host when overproduced in
cytoplasmic region
•Target protein coupled with Signal sequences
(malE, ompA ,phoA )
•Human growth hormone which accumulated
in the periplasm of E. coli was able to be
exported into the medium upon induction of
bacteriocin release protein BRP
5 Cleavage of fusion protein –
some recombinant protein
with fusion protein are toxic
to the cells
•Fusion protein has to be constructed with a
specific cleavage side. (cleavage proteins –
Thrombin, blood coagulation factor Xa,
enterokinase)
•Oligonucleotide linker
•Intein
6 Plasmid instability •Strong and inducible promoter
7 Modification of proteins for
improvement of separation
•Altering certain physico-chemical properties
of the target protein by means of sitedirected
19. Fusion protein
19
Fusion proteins - protects the cloned gene
product from attack by host cell proteases
Source - Glick
21. Cleavage of fusion protein
21
Oligonucleotide linker encoding the amino acid
sequence Ile-Glu-Gly-Arg can be joined to the
cloned gene.
Following synthesis and purification of the fusion
protein, a blood coagulation factor called Xa -
release the target protein
Source - Glick
22. 22
Additional purification
steps in order to
separate both the
protein and the fusion
protein from the
protein of interest.
This system has been
used to purify α-1-
antitrypsin and basic
human fibroblast
growth factor
Source - Glick
23. High recovery
23
Histidine-tagged protein - passed over an
affinity column of nickel–nitrilotriacetic acid -
eluted – imidazole
Greater than 90% recovery
24. 24
Human interleukin-2 gene - the marker peptide
sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
Dual function of reducing the degradation of the
expressed interleukin-2 gene product and then
enabling the product to be purified.
Single step by immunoaffinity chromatography
Bovine intestinal enterokinase
26. Secretion
26
Directing a foreign
protein to the
periplasm or the
growth medium
makes its purification
easier and less costly
Secretion into the,
Periplasm
Medium
28. Secretion into the medium
28
Gram-negative bacteria can secrete a
bacteriocidal protein called a bacteriocin into the
medium.
A bacteriocin release protein activates
phospholipase A
Cleaves membrane phosopholipids so that both
the inner and outer membranes are
permeabilized.
Some cytoplasmic and periplasmic proteins are
released into the culture medium
31. Objective
31
In this paper they deal with different factors
influencing protein maturation and export, such
as
(i) the nature of the signal peptide (OmpA or PhoA),
(ii) the role of charge distribution near the leader
peptidase cleavage site,
(iii) the influence of chaperones (GroES and GroEL),
(iv) the incubation temperature and inducer
concentration
(v) the use of lysis proteins and
(vi) fusion to a known, secreted protein (preMBP)
34. Methods
34
The plasmid-containing strains were grown in rich
medium at 37 ˚C Gene expression was induced by the
addition of 01-1 mM IPTG
Cells in late exponential growth were harvested and
washed in TE
The cell pellet was gently resuspended
After 10 min at room temperature, the suspension was
centrifuged for 5 min at 6000g.
The sucrose solution was carefully drained from the
tube and the pellet was resuspended in a same volume
of cold water
The resuspended cells remained on ice for 10 min and
centrifuged at 15000 g for 10 min at 4 ˚C. The
35. 35
The pellet was resuspended, freeze-thawed six
times and centrifuged for 5 min at 15000 g.
The pellet and the supernatant are referred to as
the membrane and cytoplasmic fraction,
respectively. All samples were resuspended in
SDS loading buffer
Analyzed by 14%SDS PAGE
Visualized by immunodetection with monoclonal
antibodies directed against IL-2.
38. Reasons
38
The signal peptides may have become buried
very soon after synthesis and were unable to
direct the protein to the export machinery
Altered the charge distribution near the cleavage
site – Site-directed mutagenesis was performed
substitution lysines (K8/K9) of IL-2 by glutamic
acid (E), the K8E and substitution of cysteine
125 (C125) by alanine 125 (A125)
Early folding or aggregation of the precursor
leads to loss translocation - chaperone factors,
GroEL, GroES, DnaK and SecB appear to be
required to prevent early protein folding
40. IL-2 bioassay
40
Biological activity of human
IL-2 was tested using the
IL-2-dependent murine T
lymphocyte cell line CTLL-2
Both recombinant proteins
were found to be active in
the assay. Interestingly,
FXa cleavage yielded
aprotein with higher specific
activity which was very
Similar to that of a
preparation of Chinese
Hamster ovary-derived
recombinant IL-2
42. Introduction
42
Human G-CSF - single chain polypeptide
containing 174 amino acid residues
(MW=18.8kDa, pI=6.1)
One of the hemopoietic growth factors which
plays an important role in stimulating,
proliferation, differentiation, and functional
activation of blood cells.
It contains a free cysteine at position 17 and two
intramolecular disulfide bonds
43. Objective
43
To development of an efficient and scalable
procedure for production and purification of
recombinant human (rh-GCSF) of E. coli
48. Conclusion
48
2.2 g lˉ¹ rh-GCSF was produced in batch
cultivation with recovery yield about 40% and with
purity over than 99%.
The process established in this study may be
functional in the recovery of other proteins
expressed in E. coli as cytoplasmic IBs.
49. References
49
1. Erwin Flaschel et.al., 1993 Improvement of Downstream
Processing of Recombinant Proteins By Means of
Genetic Engineering Methods Vol. 11, Pp. 31-78
2. http://www.biologydiscussion.com/biotechnology/downstr
eam-processing/stages-in-downstream-processing-5-
stages/10160
3. Bernard R. Glick, Jack J. Pasternak, and Cheryl L.
Patten. Molecular biotechnology : principles and
applications of recombinant DNA
4. Gabrielhea Lfmann et.al., 1993, Targeting of interleukin-2
to the periplasm of Escherichia coli, Journal of General
Microbiology, 139, 2465-2473.
5. S. Abolghasemi Dehaghani et.al., June 2010 An efficient
purification method for high recovery of Recombinant
Human Granulocyte Colony Stimulating Factor from
recombinant E.coli International Journal of
Environmental Science and Development, Vol. 1, 111-