Inducible Protein Expression in LEXSY with Monitoring Induction

Transcription

1 Inducible Protein Expression in LEXSY with Monitoring Induction - 3rd Generation Expression Kit - Cat.-No. EGE-1410 Jena Bioscience GmbH Loebstedter Str Jena, Germany Tel.: Fax: info@jenabioscience.com

2 Target gene is introduced into LEXSY host T7-TR with new generation expression vector plexsy_i-blecherry3 Case study 1 Intracellular target protein - architecture of the expression strain - T7pol TR sat hyg ssu (multiple) ssu (multiple) LEXSY host T7-TR TR provided Po X blecherry odc (singular) Target protein is co-expressed with BleCherry conferring both, antibiotic resistance for selection as well as visible fluorescence under daylight as a direct read out for protein production. 1

3 Fluorescence measurement showed homogeneity of inducible clones - reduced expression of constitutive clones #1, H- H+ S1- S A 26h 50h 74h 96h OD nm 4,5 4 3, ,5 2 1,5 1 0,5 0 OD 0h OD 26h OD B 50h OD 74h OD H- H+ S1- S h A: Cherry fluorescence* () and B: Growth (OD 600 nm ) of LEXSY clones #1-12 at h post induction [- (No Tetracycline), + (Tetracycline 10 mg/l)], H host control, S polyclonal selection in suspension culture. * Samples of 1 ml were taken and fluorescence was determined (ex 590 nm / em 620 nm) allowing i) monitoring the time course of expression for individual cultures and ii) comparison of expression levels for various cultures 2

4 Target protein expression correlated with Cherry fluorescence - demonstrated t d in Coomassie gels and in situ fluorescence scans Tet A Blecherry target B Selected LEXSY clones were cultivated as suspension culture and induced for h. Cell extracts were separated by SDS-PAGE (A) and SDS gels were fluorescence scanned in situ (B) 3

5 Target gene is fused with signal peptide coding region on expression vector plexsy_i-blecherry3 and introduced into LEXSY host T7-TR Case study 2 Secretory target protein - architecture of the expression strain - T7pol sat ssu (multiple) TR hyg ssu (multiple) LEXSY host T7-TR TR provided Po SP Y blecherry odc (singular) Secretory target protein is co-expressed with BleCherry conferring both, antibiotic resistance for selection as well as visible fluorescence under daylight as a direct read out for protein product- ion. Target protein is secreted to the mediumm whereas BleCherry remains cell bound. 4

6 Target protein was inducibly secreted from LEXSY clones Recombinant clones were pre-selected using BleCherry fluorescence associated marker and induced for protein secretion in suspension cultures secreted target protein (28 kda) Tet H #1 #2 #3 #4 NC Supernatants were 60x TCA concentrated and resolved by SDS-PAGE. Each lane represents 1.2 ml of culture, [- (No Tetracycline), + (Tetracycline 10 mg/l)]. #1-4 pre-selected clones, H LEXSY host control, NC negative control non-secreting LEXSY strain. 5

7 Inducible BleCherry expression was comparable with different targets rf fu/ml h 72h 0 H Cherry fluorescence* () of LEXSY cultures (#1-5) with different target genes 48 and 72 h post induction. * Samples of 1 ml were taken and fluorescence was determined (ex 590 nm / em 620 nm) allowing i) monitoring the time course of expression for individual cultures and ii) comparison of expression levels for various cultures [- (No Tetracycline), + (Tetracycline 10 mg/l)], H host control. 6

8 Co-expression of BleCherry reporter enables colony Screening by eye Upon induction expression clones can be identified by their cherry color at daylight and flawed clones (gray color) can be excluded from further analysis. 56/60 clones were cherry inducible 2/60 clones were cherry constitutive (#1,2) 2/60 clones were app. cherry negative (#17,18) Transfected cells were spread onto a nitrocellulose membrane on top of LEXSY BHI agar and after colonies had emerged they were transferred to BHI agar with inducer Tetracycline by colony lift technique. Cherry colour was visible ibl 24 h post induction. i In constitutive i clones cherry colour appeared before induction. 7

9 Numerous target proteins were efficiently expressed with 3rd generation of inducible LEXSY BleCherry Target 1 Target 2 b nb Tet c c H Target 3 Target 4 intracellular cell extracts secretory conc. supernatants Supernatants were TCA concentrated and resolved together with cell extracts by SDS-PAGE [- (No Tetracycline), + (Tetracycline 10 mg/l)], b boiled sample, nb not-boiled sample, H host control, c constitutive expression controls. 8

10 Appendix Broad window of clone picking for expansion of recombinant strains - Clones retained inducibility after 12d of induction on plates - large colonies small colonies large colonies small colonies early clone picking 2d post induction late clone picking 6d post induction app. cherry negative 6d post induction late clone picking 6d post induction very late clone picking 12d post induction * Samples of 1 ml were taken and fluorescence was determined (ex 590 nm / em 620 nm) allowing i) monitoring the time course of expression for individual cultures and ii) comparison of expression levels for various cultures [- (No Tetracycline), + (Tetracycline 10 mg/l)], H host control, S polyclonal selection in suspension culture. 9

11 Appendix Broad induction plateau in inducible LEXSY - Modulation of expression levels by titration of inducer at low concentrations - % EGFP ,05 0,1 0, ,01 0, Tet (μg/ml) data based on Facs analysis 24h post induction growth inhibition 10