in this pdf, we will explore the problems associated with the production of recombinant proteins and learn more about pharmaceutical biotechnology

1. Problems associated with the
production of recombinant
protein

2. Antibodies
Recombinant
DNA products
Proteins

3. Methods of
protein
classification
Chemical
composition
Shape
Solubility
Biological
function

4. Chemical
composition
Simple
protein
Conjugated
protein

5. Methods of
protein
classification
Chemical
composition
Shape
Solubility
Biological
function

6. Shape
Fibrous
protein
Globular
protein

7. Methods of
protein
classification
Chemical
composition
Shape
Solubility
Biological
function

8. 1
Determining DNA, RNA,
and protein sequence
3
2
Cloning of correct gene
into the expression vector
Transformation of vector
into host cell and
expression

9. Prokaryotic cells Eukaryotic cells
Easy to:
➢ Grow
➢ Genetically manipulated
Not all genes are able to be expressed in
prokaryotic cells
Antibiotic resistance genes for increased selectivity
of transformed bacteria
Has all necessary promoters and terminators in
gDNA already
Lack of before and after translation protein
modification pathways for correct protein
manufacturing

11. Problems associated with the production of
recombinant protein
Loss of expression
• Plasmid-Based Systems
• Chromosomal Integration
• Viral vectors
Posttranslational
processing
• Folding, aggregation &
solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational
Modifications
Transport and
Localization

12. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors

13. G.R: Loss of expression?
• expression can be lost due to:
➢structural changes in the recombinant gene
➢disappearance of the gene from host cells
Gene of interest
In Plasmid
delivered by a virus
integrated into the host’s chromosome

14. Plasmid-Based Systems
• Plasmid:
➢extra-chromosomal self-replicating cytoplasmic DNA
elements
➢found in prokaryotes and eukaryotes.
➢Used as molecular vehicles for recombinant genes
➢most popular choice when using prokaryotes as hosts?
▪ as genetic manipulation of plasmids is easy

15. Formation of a single dimeric circle with 2 ori
• As plasmid copies have the same sequence, they can recombine
and form a single dimeric circle with two origins of replication.
This results in fewer independent units to be segregated between
daughter cells, and consequently plasmid loss can increase

16. Plasmid segregational instability
Plasmid loss is the main cause
of reduced recombinant protein
productivity in plasmid-based
systems

17. Metabolic load and plasmid
• the size of the insert
• temperature,
• expression level
➢Constitutive gene expression may increase plasmid instability because
the metabolic load of recombinant protein production is constantly
present.
• recombinant protein yield
• toxicity of the expressed protein toward the host

18. Gene dosage
• refers to the number of copies of a specific gene present within an
organism's genome
• Gene dosage is higher than when the recombinant gene is
integrated into the host’s chromosome
• Medium to high copy number plasmids:
➢Relaxed replication
➢Random distribution
➢Relatively low loss: Continuously growing and toxic genes/gene products
will lead to plasmid loss.

19. How to increase gene dosage in E.coli
genome?
Method How does is work? Limitations
RecA duplication of insert (Olson
et al. 1998)
• By RecA protein, which plays a
crucial role in homologous
recombination to duplicate the
insert
• Using this method leads to an
increased gene dosage.
• The reported range of copies
was 15 to 40.
• unstable without a selectable
marker.
Tn1545 site-specific
recombination (Peredelchuck
and Bennett 1997)
• site-specific recombination
mediated by transposon
Tn1545.
Time consuming

20. Plasmid copy number
For applications such as DNA
production for gene therapy, high
plasmid copy number is an
important objective function

21. Plasmid copy number
• copy-number control genes regulate plasmid copy-number.
• Plasmid copy number is an inherent property of each expression system
• Plasmid copy number depends on:
➢ the number of plasmid copies at the time of cell division and their random distribution
between daughter cells
➢The host
➢Culture conditions
• How to ensure plasmid survival in the cell population?
• low-copy-number plasmids guarantee their persistence by multimer
resolution through:
❑ site-specific recombination systems (cer sequence)
❑active partition mechanisms, such as the par sequences.

22. Although high plasmid copy numbers are generally desired protein
yield, this might not always be true. G.R?
• Because it may drive for improving recombinant
high protein production rates, which can result in:
• protein aggregation
• Deficient posttranslational modification.
• Reduced translation efficiency despite high
plasmid copy number
Low recombinant protein
yields can also occur in
cells with a high plasmid
copy number:
• Reduced Translation
Efficiency
• Metabolic Burden
• Cell Stress

23. Plasmid structural instability
A complete elimination of
recombinant protein
production
accumulation of aberrant
recombinant proteins with
minor changes in the original
amino acid sequence
(deletions, additions, or
substitutions)
To
Solve
this we
preform

24. Plasmid structural instability
complete amino acid
sequencing of the
recombinant protein
DNA sequencing of the gene
of interest
To
Solve
this we
preform

25. Increased Plasmid stability by using
selectable markers
Genes for
antibiotic
resistance
Complementation
Genes or repressors
that lead to cell
death upon plasmid
loss.

26. Antibiotics are expensive, and their presence is
undesirable in food and therapeutic products
deletion of an essential
gene from the bacterial
chromosome and its
inclusion in the plasmid
the introduction of a growth
repressor in the bacterial
genome and its antidote in
the plasmid
To
Solve
this we
preform

27. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors

28. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors

29. Chromosomal integration
• A powerful alternative for overcoming problems of expression
stability in plasmid-based systems
• suitable for metabolic engineering of the host

30. Chromosomal integration Vs. Plasmid-
Based Systems
ADVANTAGES DISADVANTAGES
Reduce metabolic burden Labor intensive
Stable inheritance Time consuming
Improved genetic stability Low copy number
Reduced risk of Horizontal Gene Transfer (HGT) Integration of gene of interest into an inactive region
of chromosome
Long term expression
The recombinant cells obtained are able to grow in
the absence of antibiotics without any reduction of
recombinant protein yields.
To overcome
this
use of locus control
regions (LCRs), which
ensures transcriptional
regulation of the
transgene.

31. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors

32. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors

33. Viral vectors
• Easy and very effective way of delivering the gene of interest
• Viruses have evolved to deliver their genetic material to the host in an
efficient and non-destructive way.
• Useful for production in higher eukaryotes because of its simplicity
• Retroviruses: promote integration of the viral genome into the cell’s
chromosome.
• Viral expression systems are a niche for industrial protein production.
• Ex →(BEVS):
➢utilized to commercially produce several recombinant proteins.
➢suitable for the production of vaccines.
➢application for viral vectors is gene therapy.
the insect cell
baculovirus
expression
vector system

34. Viral vectors
• transient expression:
➢ refers to the temporary expression of genes in the host organism for a
limited duration
➢Allow rapid production of proteins or other gene products without the
need for stable integration into the host organism
➢utilized for rapidly generating sufficient amounts of protein for laboratory-
scale applications or preliminary testing of drug candidates
Once a promising molecule is identified, a stable cell line can be generated

35. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors

36. Problems associated with the production of
recombinant protein
Loss of expression
• Plasmid-Based Systems
• Chromosomal Integration
• Viral vectors
Posttranslational
processing
• Folding, aggregation &
solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational
Modifications
Transport and
Localization

37. Problems associated with the production of
recombinant protein
Posttranslational processing
• Folding, aggregation & solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational Modifications

38. Post-Translational Modifications
(PTM)
the extent of modification depends on the host utilized, being the
modifications performed by higher eukaryotic cells closer to
those found in human proteins

39. Folding, aggregation and solubility
Foldases Chaperons
Key player molecules of
folding
Inclusion bodies → misfolded
proteins that accumulate in
intracellular aggregates

40. Example on human pathologies characterized by
intracellular protein aggregation and accumulation
Alzheimer’s
disease
Parkinson’s
disease
Huntington’s
disease

41. Folding, aggregation and solubility
• One of the main causes of incorrect protein folding is cell stress,
which may be caused by:
➢ heat shock
➢nutrient depletion
➢or other stimuli
• How do cells respond to stress?
• by increasing the expression of various chaperones, some of them of the
hsp70 and hsp100 families.

42. Strategies to reduce aggregation protein
engineering
Changing the
extent of
hydrophobic
regions
Using fusion
proteins

43. Strategies to reduce aggregation protein
engineering
Using fusion
proteins
• Recombinant fusion proteins are created artificially
by recombinant DNA technology for use in biological
research or therapeutics.
• The purpose of creating fusion proteins in drug
development is to impart properties from each of
the "parent" proteins to the resulting chimeric
protein.
• Fused proteins often contain a peptide native to the
host used. For example, fusing single chain
antibodies to an E. coli maltose-binding protein
allows the production of soluble functional protein
in E. coli cytoplasm

44. Problems associated with the production of
recombinant protein
Posttranslational processing
• Folding, aggregation & solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational Modifications

45. Problems associated with the production of
recombinant protein
Posttranslational processing
• Folding, aggregation & solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational Modifications

46. Proteolytic Processing
• Proteolysis: breakdown of proteins into smaller
polypeptides or amino acids through the hydrolysis of
peptide bonds by a protease.
• Signal peptides: needed to direct protein to the various
cellular compartments Signal peptides
must be cleaved?
To obtain a
functional protein

47. • Endoproteases or endopeptidases: proteolytic peptidase that
break peptide bonds of nonterminal amino acids (within the
molecule)
• There is some protein smust be expressed as proproteins
because prodomains act as folding catalysts → cells utilize
endoproteases to produce the mature active protein.
• Example:
➢Proteases
➢Insulin
➢penicillin acylase

48. Removal of the N-terminal methionine
• occurs only in proteins in which the second amino acid is:
➢ alanine
➢Glycine
➢Proline
➢Serine
➢Threonine
➢Valine
• This processing is performed by a methionine aminopeptidase
(MAP)

49. Reduction of yield of secreted proteins
To
Solve this
Overxpression
E.Coli signal
peptidase I
Bacillus subtilis
peptidase I

50. Glycosylation
• Complex form of protein modification occurring in the secretory
pathway
• requires several consecutive steps
• involves tens of enzymes and substrates.
• It usually occurs in the endoplasmic reticulum and Golgi
apparatus of eukaryotic cells
• N-glycosylation has been detected in proteins produced by
bacteria

51. Glycosylation
• In many cases, glycosylation determines protein:
➢ stability
➢Solubility
➢Antigenicity
➢Folding
➢Localization
➢biological activity
➢circulation half-life.

52. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation

53. 3 types of glycosylation
• the most studied and is
considered as the most relevant
for recombinant protein
production.
• N-glycans linked to an
asparagine (Asn) of consensus
sequence
Asn – X – (Ser/Thr)
N-glycosylation

54. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation

55. 3 types of glycosylation
• O-glycans linked to oxygen atom of:
➢Serine (Ser)
➢Threonine (Thr)
O-glycosylation

56. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation

57. 3 types of glycosylation
• Attachment of glycans to
trptophan
• C-linked glycosylation has
hardly been studied and
little is known about its
biological significance .
C-glycosylation

58. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation

59. Glycosylation

60. Glycosylation
• Glycosylation profiles are protein-, tissue-, and animal specific .
Nonauthentic glycosylation → may trigger immune responses
when present in proteins for human or animal use.
• Therefore, authentic glycosylation is especially relevant for
recombinant proteins to be utilized as drugs.

61. Problems associated with the production of
recombinant protein
Loss of expression
• Plasmid-Based Systems
• Chromosomal Integration
• Viral vectors
Posttranslational
processing
• Folding, aggregation &
solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational
Modifications
Transport and
Localization

62. • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC316774/
• Proteolytic Post-translational Modification of Proteins: Proteomic
Tools and Methodology - PMC (nih.gov)