PRODUCTION OF SERINE PROTEASE ISOLATED

FROM THERMOASCUS AURANTIACUS VAR

LEVISPORUS USING PICHIA PASTORIS

Nur Okyay
Hande Doan
Ezgi Tanl

Aim
Large scale production of serine
protease
Active
Thermostable
Low cost
Low contamination risk
High yield

Serine Protease
Enzyme cleaves peptide bonds in
proteins
Found in both eukaryotes and
prokaryotes
Named after as nucleophiles to
attack the peptide bonds (cysteine,
serine and threonine).
Used in : detergent, meat and
brewing industry.

Thermoascus aurantiacus
Thermophilic fungus
Ability to secrete large amounts of
thermostable enzymes
Used as platform for the production
of thermophilic
enzymes

Pichia pastoris
species ofmethylotrophicyeast
uses methanol-methane as carbon source

Advantage
reduces contamination risk in production
Produces extracellular protein
Easier to separate/purificate

Production of active proteins

Higher yield than other expression systems
Lower cost

Project Summary
Cloning serine protease gene from T.
aurantiacus E.coli DH5 strain
Selecting for transformants using
Amp(R) and Blue-White Screening
Transforming isolated plasmids from
E.coli to P. Pastoris
Screening and protein activity analysis
Large scale production, separation and
purification of serine protease

1. Isolation and Amplification of Serine

Protease Gene from T. aurantiacus
T. Aurantiacus : mRNA donor
Total RNA isolation
cDNA template : RT-PCR
Primers: (dizayn)

Resource: Thermoascus aurantiacus var. levisporusserine proteasemRNA,

complete cds [http://www.ncbi.nlm.nih.gov/nuccore/EU364816.1]

2. Cloning to E.coli Selection and

Screening

Resource: http://kr.sinobiological.com/Vector-pMD18-T-Simple-a-

 Vector is cut using EcoR V

Amplified cDNA template is cut with
EcoR V too
The vector and insert is ligated.
Organism: E.coli DH5
Method of Producing Competent
Cells: Electroporation

 Selection of transformants: Ampicillin

resistance
Added to blue-white screening plates
Non-transformants die

Selection for recombinants: BlueWhite Screening

Vector contains lacZ gene
Orientation
Non-recombinants will die.

3. Transforming P. pastoris
Plasmid isolation from organisms in
blue colonies.
Cutting the insert using EcorV
Amplification of insert: PCR
Primers :
5'-CCGGAATTCTCACCCGTCGTCGTAGACTCGAT3
5'TTGCGGCCGCCTACGCAGTAACAGCATCCTTGA
T-3

Designed with EcoRI and NotI restriction

Resource: http://www.genesou.com/?product=ppic9k

 Expression Vector: pPIC9K

Used for expression in P. Pastoris
Contains alcohol oxidase genes : Aox1-Aox2

Vector and insert are cut with EcoRI

and NotI.
Vector and insert are ligated.
The vector is linearized with XbaI
The cells are made competent using
electroporation (artlar)

4. Selection and Screening (P.

Pastoris)
Incubation (media nn zelliklerini
yaz)
Screening method: EasySelect
Pichia Expression kit (Invitrogen)

[http://tools.lifetechnologies.com/content/sfs/manuals/easyselect_man.pdf]

5. Separation and purification of

protease
Purification of protease:
0-4C
Method: Ammonium sulfate precipitation
(70% saturation)
Centrifugation : collect the precipitates
Gel Filtration: to remove ammonium
sulfate
Sephadex G25

Molecular weight of protein : 59.1

kDa

Ammonium Sulfate Precipitation: [http://portal.unimap.edu.my/]

Gel filtration [http://www.ucl.ac.uk/~ucbcdab/enzpur/amso4.htm]

6. Protease Activity Analysis

Spectrophotometric Analysis
Substrate : Casein
Protease digests casein. Tyrosine amino acids are released.

Reagent: Folin & Ciocalteus Phenol

Form complex with tyrosine
Give blue colored product (product adna bak)

Spectrophotometer
Blue colored product is detected
Absorbance values are compared to standard (known
tyrosine-reagent complex concentration)

According to comparison, the colony that gives

maximum yield is selected to use in large scale
production.

7. Large Scale Production

Bioreactor: Stirred tank fermenter
1st cultivation: Glycerol complex
medium
To generate biomass in a short time

Mass production
Volume: 30 L
Working volume: 24 L
Impeller Speed: 100-1370 RPM
Temperature : 30C

Parameter

Constraint

pH

5.50

Methanol solution concentration

YTM 4.4% (w.w) + methanol

96.6% (w/w)

Final methanol concentration

2 g per litre

Growth media components

4.6 g magnesium sulphate

heptahydrate, 95.2 g glycerol, 0.4
mg biotin, 4.56 g yeast trace
mineral (YTM) solution, 9.4 g
potassium dihydrogen sulphate,
0.28 g calcium chloride
dehydrate, and 15.7 g ammonium
sulphate

YTM components

46.3 mg/l boric acid, 9.2 g/l

sulphuric acid, 760.6 mg/l
manganese sulphate, 12g/l ferric
chloride hexahydrate, 5.0 g/l zinc
sulphate heptahydrate, 207.5
Reference: Gurramkonda C., Polez S., Skoko N, Adnan A, Gbel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky
mg/lto potassium
iodide
and
484
S, and Rinas U. 2010. Application of simple fed-batch technique
high-level secretory
production
of insulin
precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Fact.
mg/l sodium molybdate
2010; 9: 31.

 The growth media and organisms are

placed in sterile bags, then the bags
are placed into fermenter one by
one.
Allows harder agitation
Decrease separation-purification time
Bags contain pores for nutrient, pH
arrangements etc.

8. Purification
Ion Exchange Chromatography
Proteins are charged molecules.
Serine protease : positively charged

Negatively charged molecules are attached to

solid stationary phase
Sample is in liquid mobile phase
Charged proteins interact with negatively
charged ions at the stationary phase while flow
of mobile phase
After flow, protein molecules are eluted from
the stationary phase.

DEAE Sepharose Flow: https://www.gelifesciences.com/gehcls_images/GELS/Related

%20Content/Files/1314742967685/litdoc71500964_20141204211833.pdf

9. Freeze Drying and

Packaging
Purified samples are freezed at -80C
for at least one night.
Freezed liquid is sublimied using cold
vacuum.
Powder enzyme are weighed and
packaged.