generation of recombinant collagenases class i & ii. a new ... · prof. giulio ghersi,...

Prof. Giulio Ghersi, Dipartimento di Scienze e TecnologieBiologiche Chimiche e FarmaceuticheViale delle Scienze, ed 16 – Palermo.

Vice Presidente ATeN CenterResponsabile polo CHABViale delle Scienze, ed 18 – Palermo

Presidente ed ADABIEL S.r.l.Via del Mare 3, Campobello di Mazara (TP)

Generation of recombinant collagenases class I & II. A new approach in cell

purification for research and clinical applications

Summer School in Advanced Biotechnologies 2017 –September 3-6, 2017 SION (CH)

Clinical Hurdles in

Cell Transplantation

The need for standardized enzymes


The solution: Recombinant collagenases


Col_I_Optimizer_Ver4 (3053 nt) [ORDINATO BLUE Heron]

ccatgggatccatgatcgcgaacaccaatagtgagaaatacgactttgaatacttgaacggtctgagctacacggaactgactaacctg

atcaaaaacattaagtggaaccagatcaacggcctgttcaattattctactggctctcagaaattcttcggtgacaaaaaccgtgtaca

ggcgattatcaacgccctgcaggaatctggccgcacttataccgctaacgacatgaaaggcatcgagaccttcactgaagttctgcgtg

cgggtttttatctgggctactacaacgacggtctg......................tcgccactacgtcttcatttacaaacatgatt

ctgcctcgaacattagctattcactcaacatcaaaggtctgggtaacgaaaagctgaaagaaaaggaaaataacgattcttccgataaa

gcaaccgtgattccgaactttaacaccactatgcaggggtcgctgctgggtgacgattcccgcgattattactccttcgaagtaaaaga

agagggcgaagtgaacatcgaactggataaaaaagacgaatttggtgttacctggacgctgcacccggaatctaacatcaacgaccgta

tcacctatggccaggtggacggtaacaaagtttccaacaaggtcaaacttcgcccgggcaaatattatctgctggtctacaagtattct

ggatctggtaattacgaactgcgtgttaacaagtaagagctcaagctt

Optimized using Optimizer

COLH V5 (2970 bp) Cloning in pCR2.1 or pBluescript(without its promoter, to prevent

toxicity in E. coli)

ggatccaccATGGTTCAAAACGAAAGCAAACGTTACACCGTGAGCTATCTGAAGACCCTGAATTACTACGACCTGGTAGATCTGCTGGT

CAAGACGGAAATCGAGAACCTGCCGGACCTGTTCCAGTATAGTAGCGATGCCAAAGAGTTTTACGGGAACAAAACGCGCATGTCGTTCA

TTATGGATGAAATCGGTCGCCGTGCCCCGCAGTATACGGAAATCGATCATAAAGGGATTCCTACTCTGGTAGAAGTGGTCCGCGCTGGG

TTTTATCTGGGGTTTCACAATAAAGAACTGAATGAAATTAACAAGCGTAGTTTTAAGGAGCGCGTGATTCCAA................

............TGAAAATACCAGCGACCAGGACTATTTTTATTTTGATGTTATTACCCCAGGTGAGGTTAAAATTGACATTAACAAAC

TGGGCTACGGCGGCGCCACCTGGGTCGTGTATGATGAAAATAATAACGCGGTGAGCTACGCGACCGATGACGGGCAGAACCTGAGCGGC

AAATTCAAGGCCGATAAACCGGGCCGCTACTATATTCATCTGTATATGTTTAACGGCAGCTATATGCCGTATCGCATCAATATCGAAGG

CAGCGTGGGCCGCtaactcgagaagctt

Optimized with GENEius, codon usage of E.coli (semi HEG)

Class I - Collagenase G : optimized sequence (3053 nt)

Class II - Collagenase H - optimized sequence (2970 nt)




Optimization by Codon Usage Escherichia coli K12



Host: E. coli

• TOP10 (contain the gene Bla for the resistance to AMP)

• F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1

araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-

• BL21AI (induction by arabinose of T7-pol gene; absence of endogenes

proteases; contain the gene Tet to be resistant to tetracicline) Transformed

plasmids containing T7 promoter driven expression are repressed until L-

arabinose induction of T7 RNA polymerase.

• F– ompT gal dcm lon hsdSB(rB- mB

-) araB::T7RNAP-tetA



1000 bp

1500 bp

2000 bp

3000 bp ~3050 bp~3170 bp

Collagenasi G3050bp

M

Sequences codifying CollG and Coll H were optimized by silica application using the Codon Usage Database (http://www.kazusa.or.jp/codon) to be efficiently expressboth in the E.coliBL21A1 clone (which constitutively low express proteolytic enzymes).

Optimized sequence in “transport” plasmid.

Generation of chimera recombinant C. Hystolithicum collagenase G (Coll G) and H (Coll H), full length forms, in E.coli.



1000 bp

1500 bp

2000 bp

3000 bp

~6700 bp

M C2 P2



Chimera MBP-Collagenase Coll G (C. histolyticum)

Chimera MBP- Collagenase Coll H (C. histolyticum)

CommonsegmentCatalytic domain

Zn2+

H2N COOHPre-peptideCollagen binding

domainCollagen binding

domainMaltose Binding Protein

H2NCommonsegmentCatalytic domain

Zn2+

COOHPre-peptideCollagen binding

domainMaltose Binding Protein Common

segment



205

116

97,4

66

45

29

205

116

97,4

66

45

29

IPTG ( ) ( ) ( ) ( ) _ _ + +

mock mock mock mock Rec.Rec.Rec.Rec. M M

S INS

Expression by IPTG induction and proteins solubilization



M = Molecular weight Markers; 1, 3, 5 and 7 = IPTG non Induced cells; 2, 4, 6, and 8 = IPTG Induced cell. 1, 2, 5, and 6 = Comassie blue staining; 3,4, 7 and 8 = gelatin zymography.

Coll G and H expression in E. coli BL21AI



M ML LUB UB BB

M = MarkersL = LysateUB = UnboundB = Bound

205

116

97,4

66

45

29

_

__

_

_

_

Coll G Coll H

Affinity chromatography purification of Chimera/Recombinant enzymes


The solution: Fermentation production and scale up

• closed system

• no control on bacterial growth rate μ (h-1 )

• only PH, temperature and stirring speed can be regulated

Batch fermentation



• 6 L volume•10 % DO saturation• 1 M IPTG Single shot induction

• 18 OD final concentration• 2 gr. of col G• 1.8 gr.of col H

Batch fermentation



Batch fermentation output:• Final OD of 22 units• 6 gr. of col G and col H produced per experiment

6 L Batch fermentation Multiple shots induction system, DO 20%



Fed-batch fermentation

• Exponential feeding strategy

• Costant μ ( h-1 )

• Multiple shots induction system



Materials and methods:• 4 liters starting volume•800 ml of N feed solution•200 ml of P feed solution•200ml of C feed solution (glycerol)•3ml/min N/P feed pump speed•1,5ml/min C feed pump speed•Exponential growth method (pumps speeds increases in correlation with cell concentration increase)•Proportional multiple steps induction system

By applying a simple mass balance equation, it was possible to evaluate the right amount of each of three basic elements (C, N, and P) essential

for our recombinant bacteria growth.

Fed-batch fermentation



• Final cell concentration: 34 OD• 8,2 gr of purified col G and 7,2 gr of purified col H per experiment

4 L Fed-batch fermentation output



•64 OD units per fermentation• 12,5 gr of Col G and 12,1 of Col H purified by each production

8 L animal free medium fed-batch fermentation grow curve



Downstrem purification processing



Up to 98%

Col G purified enzyme on Size Exclusion HPLC - purity evaluation


Roche Serva

Wunsch Units

Vitacyte Sigma

FALGPA Units

Sigma Worthington

CDU

Abiel

Grassman Units

Measured activity: class I + class II + proteasesVariability of class I:class II ratio. batch-to-bacth variation.!

COLLAGENASES

ACTIVITY

SYNTHETIC

PEPTIDES

NATIVE

COLLAGEN

WUNSCH FALGPA GRASSMAN CDULABELING

WITH 3H

CII >>CI CI >CIICII >CI

Remember: Units are not easily comparable, especially if you compare a mix of enzymes and a pure collagenase.


Quantification of collagenase classes a critical step


Quantification of collagenase classes a critical step

The Applications: Preclinical applications

Human Langherans’ islets

hMSC from adipose tissue

Murine Langherans’ islets

Osteoblasts for bone regeneration

Fibroblasts for dermis regeneration

Hepatocytes for liver regeneration and BAL

Condrocytes for cartilage regeneration

Murine MSCs from adipose tissue

Pre-clinical studies to validate COL G and COL H enzymatic activity and efficiency


The Applications: Preclinical applications


Puridied isletson dencity gradient system

Prepurification Islets from rat pancreas stained with dithizone

Enzymatic Digestion using recombinant enzymes

Optimization of Islets from rat pancreas in extraction/purification and storage/transport procedurres.


Summer School in Advanced Biotechnologies 2017 –September 3-6, 2017 SION (CH) R

elea

sed

insu

lin (u

g/li

ter)

15

30

45

60

Time from induction (min)

15 30 60

Figure 3: Islet insulin response to glucose

induction after 1 week from isolation

Using a calibrate mix of class I (Col G) and class II (Col H) collagenases together thermolysin in defined proportion of enzymatic activities (New patent submission February 2016) we obtain 1.250 +/- 250 IEQ from a pancreas of Wistar rat having a weight of 150-180 gr.


0"50"

100"150"200"250"300"350"400"450"

Rat$D5$glycemia$evalua0on$before$and$a7er$islets$transplanta0on$$

Glucose"m

g/dl"

Enzymatic Digestion using recombinant enzymes

Extraction of functional adult cardiomyocytes from rat heart

1 day

a-actinin Troponin Merge + DAPI

a-actinin Troponin Merge + DAPI

1 day

10 days

Monica SalamoneLorenzo VolpeAlessandra Lo CiceroSilvia RigogliusoSimona Campora

Giorgia AdamoLucrezia Pinnavaia


Grazie

Thank You

generation of recombinant collagenases class i & ii. a new ... · prof. giulio ghersi,...

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