Production of proteins from cloned genes

1
????? ??????? ?? ?? ??? ???? ??????? ????????
??? ? ????? ????? ?????? ???????? ????
??????
2
Major topics to be covered

• Expression in E. coli
• Problems with the production of recombinant
  proteins in E. coli
• Production of recombinant proteins by eukaryotic
  cells

3
Culture methods for biotech microoganisms

• Batch culture
• Continuous culture (fermentor)

4
The technique not limited to antibiotics,..
5
(No Transcript)
6
(No Transcript)
7
Production of animal protein by bacteria
8
The technique continued to gene cloning for
production of compounds not possible by previous
means

• Low production or,
• High yield production of recombinant proteins

9
Problems with production of recombinant proteins
in E. coli

• Problems resulting from sequence
• Introns
• Presence of possible terminations for E. coli
• Codon bias of the foreign gene
• Problems caused by E. coli
• Posttranslational modification
• Protein folding
• Degradation of foreign protein

10
(No Transcript)
11
Special vectors for expression of foreign genes
in E. coli

• For a good expression vector three most important
  signals must be considered, promoter is the most
  critical one

12
Exploitation of an expression vector, to achieve
expression of foreign gene in E. coli
13
consensus
Pribnow box
14
Choosing promoter

• Efficiency of expression
• Strong promoters
• Weak promoters
• Regulation of promoter
• Induction
• Repression

15
(No Transcript)
16
(No Transcript)
17
Examples of promoters used in expression vectors

• lac promoter
• trp promoter
• tac promoter
• lpL promoter

18
Production of recombinant proteins by eukaryotic
cells

• Yeast and filamentous fungi
• Insect cells
• Plants
• Animal cells
• Pharming

19
Saccharomyces cerevisiae

• Most popular microbial eukaryotic for production
  of rec. proteins
• Often GAL promoter is used (galactose epimerase
  promoter), regulated by galactose level in medium
• PHO5 promoter is regulated by phosphate level of
  medium
• CUP1 is induced by copper

20
Benefits of yeast Saccahromyces cerevisiae

• Yield is high
• S. cerevisiae is a safe organism
• Known biochemistry and genetics

21
Disadvantages of yeast Saccahromyces cerevisiae

• Incorrect glycosylation (hyperglycosylation)
• No efficient secretion in medium
• Codon bias is still a problem

22
Other yeasts and fungi

• Pichia pastoris (30 total cell protein, less
  glycosylation, less inmmunogenicity), uses AOX
  promoter induced by methanol
• Hansunella polymorpha
• Yarrowia lipolitica
• Aspergillus nidulans, uses glucoamylase promoter
• Trichoderma ressei, uses cellobiohydrolase
  promoter

23
(No Transcript)
24
(No Transcript)
25
Animal cells

• They require solid surface to grow (mostly)
• Rate of growth and maximum cell densities are low
• The promoter is normally derived from a virus
  (SV40)
• Protein processing is correct
• Expensive
• Requires rigorous control (safety about viruses,
  purification,..)

26
Insect cells

• High yields (50 of total cell protein)
• No correct glycosylation

27
Pharming (transgenic animals)

• Expensive to create
• Genetic inheritance is possible (advantage)
• Beta lactoglobulin promoter is used to secret
  product in milk
• Easy purification (due to secretion)
• Correct modification

28
Transgenic animals
29
(No Transcript)
30
Pharming
31
Plant cells

• Correct post-translational modification
• Plants can be grown to high densities
• Tubers or fruits (storage organs) are rich in
  protein
• Cheap and low technology is used

32
Cloning of IFN alpha-2 in a pET plasmid
33
Some recombinant proteins approved for clinical
use

• Name Application

Human Insulin Diabetes Interferon alfa2 Hairy
cell leukemia Beta Interferon Multiple
sclerosis Gamma interferon Antitumor
agent Tissue plasminogen activator Acute
myocardial infarction Human growth
hormone Pituitary dwarfism Erythropoietin Anemi
a associated with renal failure Interleukin-2
Cancer Interleukin-4 Cancer, vaccine
adjuant Hepatitis B surface antigen Vaccination
against HBV Granulocyte stimulating
factor sepsis, nutropenia Factor VIII
IX Haemophilia A B
34
The expression vector

• The pET vectors
• -Description
• -Transcriptional signals
• -Translational signals
• -Fusion tags
• -Antibiotic resistance

35
(No Transcript)
36
(No Transcript)
37
Human Chromosomal DNA extraction
38
Digestion of Chrom. DNA with BamH I
39
Plasmids pUC119 and pGEM3Z extracted from DH5a
40
Plasmids pET21b, 40b and 41b extracted from BL21
41
Direct PCR of IFN alpha coding sequence with
specific primersHC1 5gtCAGCATATGGATCTGCCTCAAAC
CCgt3HC3 5gtCGCGGATCCTTATTCCTTACTTCTTAAACTgt3
42
Digestion of PCR Amplicon with Pvu II
43
Digestion of pET21b and IFN alpha2 with Nde I
and Bam H I
44
Transformation of the ligation into DH5a strain
45
Analysis of the plasmids
46
BamHI
HindIII
f1 ori
IFNa
bla
pETHA 5916 bp
lacI
ori
47
Insert DNA sequence

• GATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCC
  TGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACAT
  GACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCTGA
  AACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTTCA
  GCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTC
  TACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACA
  GGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGG
  CTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAA
  TACAGCCCTTGTGCCTGGGAGGCTGTCAGAGCAGAAATCATGAGATCTTT
  TTCTTTGCCAACAC

48
Insert amino acid sequence

• D L P Q T H S L G S R R T L M L L A Q M R R I S L
  F S C L K D R H D F G F P Q E E F G N Q F Q K A E
  T I P V L H E M I Q Q I F N L F S T K D S S A A W
  D E T L L D K F Y T E L Y Q Q L N D L E A C V I Q
  G V G V T E T P L M K E D S I L A V R K Y F Q R I
  T L Y L K E K K Y S P C A W E A V R A E I M R S F
  S L P T

49
Pairwise alignment analysis

• BlastN
• Fasta

50
Published work

• Link to the paper