Expression Systems for Peptide Production
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1 Expression Systems for Peptide Production Susanna Leong School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore CBAS, July 2007
2 (Source: Lonza Ltd., Basel, Switzerland)
3 Challenges in Peptide Production - Demand for peptides to be produced in >100 kgs (industrial). Table 1: Peptide therapeutics dosage requirement (BioScan).
4 Challenges in Peptide Production - Demand for peptides to be produced in >100 kgs (industrial). - Right strategy of synthesis applicable on all scales. - Quality of peptides. - Downstream processing and isolation. - Economics/cost-of-goods.
5 Peptide Production Routes 2 main technologies available for peptide production: (i) Solid Phase Synthesis (ii)recombinant Technology Issues with Solid Phase Synthesis - Expensive raw material, - Complex impurity profile, - Increasing peptide lengths can lead to chain aggregation, truncated peptides, difficult purification, low yield.
6 Recombinant Peptide Production Key advantages: - Relatively cheap and simple to perform. - Easy manipulation of expression host. - Potentially higher yield at a cheaper cost (with bioprocess-centered peptide design, optimised downstream processing). Solid phase Synthesis Recombinant Technology Raw Materials: Production Cost Ratio 35%:65% 5%:95% Cost-determining Factors Raw materials, Waste streams. Unit operations, Scale-up, development costs. (Source: Lonza Ltd., Basel, Switzerland)
7 Recombinant Peptide Production Host System Product Localisation Suitable for Advantages E. coli Intracellular Hydrophilic proteins. No 2 or 3 structure. High product concentrations (5-10 g/l culture) E. coli Periplasmic Proteins with 2 or 3 structure (S-S bonds). Use of secretion to generate biological active conformations P. Pastoris Intracellular or Fermentation medium Hydrophilic and slightly hydrophobic proteins. With or without 2 or Direct secretion minimise unit operations. 3 structures.
8 E. coli Vectors for Peptide Expression - pet: high expression levels and tight control. - paed - pgex
9 Peptide expression in E. coli Stability of peptides enhanced if: (i) fused to a carrier protein, OR (ii) linked together as a large tandem polymer of repeated units. - Low expression levels, - Multi-a.a. encoding linkers for ligation of tandem repeats.
10 Tandem Peptide Fusion Protein - KSI-Peptide-His Tag Fusion (Kuliopulos and Walsh, 1994)
11 Table 2: Production of recombinant KSI-Peptide-His Tag Fusion Proteins (Kuliopulos and Walsh, 1994).
12 Fusion Protein System for short S-S containing Peptides (Fairlie et al., 2002) SS HIS SHP2 Met Peptide Fig. 1: Intracellular phosphatase (SHP2) fusion construct (Fairlie et al., 2002). Key advantages of SHP2 as a fusion partner: (i) Expressed at high levels in E. coli, (ii) Easily purified via a hexa-histidine tag, (iii) Highly soluble in native buffers, (iv) Contains a unique Met residue to facilitate cleavage. Purified peptide yield: mg/l (yield variable with peptide sequence).
13 Fusion Protein System for Small Partially Structured (Stable) Peptides (Bi et al., 2006) Fig. 2: Schematic representation of the expression vector for NTL-9-FXa-peptide (Bi et al., 2006). Key advantages of NTL (N-term domain of L9) as a fusion partner: (i) Expressed at moderately high levels in E. coli, (ii) Very soluble and highly stable (melting mid-point of 77 deg C and remains fully folded from ph 1-11), (iii) Easy purification on ion exchange (protein is very basic). Purified fusion protein yield: 70 mg/l Purified peptide yield: ~ 30 mg/l
14 Table 3: Typical expression and purified peptide yields for E. coli. E. coli (secreted) Fusion Protein Expression Yield (mg/l) Purified Peptide Yield (mg/l) E. coli (IB)
15 Yeast Host Systems Pichia pastoris (P. pastoris) - Utilises methanol as sole C source (methylotrophic). - AOX1 promoter: (i) production of alcohol oxidase for methanol oxidation, (ii) over-expression of protein genes. - Growth to high cell densities on inexpensive, defined media (Typical growth levels: >30% of total protein). - Foreign protein expression levels range from mg/l to g/l. (10 to 100 times higher protein expression levels than S. cerevisiae). - Expression levels depend on a.a. sequence, tertiary structure, expression site.
16 P. pastoris Vector for Peptide Expression - Allows intracellular expression or secretion into growth medium. secretion signal sequence from S. cerevisiae α-factor Fig. 3: Expression vector ppic9- GBP (Koganesawa et al., 2002).
17 Table 4: Effect of signal sequences on secretion levels and activities of recombinant xylanases produced in P. pastoris (Korona et al., 2006) Other leader peptides (e.g., signal sequences from S. cerevisiae secretory proteins) which enhance secretory yields: (i) invertase, (ii) acid phosphatase, (iii) killer toxin.
18 - Secretion efficiency of yeast cells influenced by: (i) Host strain and product characteristics, (ii) Signal/leader sequence, (iii) Promoter strength, (iv) Expression vectors, (v) Chaperon availability, (vi) Environmental factors (bioreactor operations, media composition).
19 Saccharomyces cerevisiae (S. cerevisiae) - Galactose-inducible promoter (GAL) most efficient, compared to constitutive promoters (e.g., TPI, ADHI). - GAL: induced by galactose, repressed by glucose. Advantage over P. pastoris: - Have stable multicopy vectors (e.g., episomal plasmids) no screening of multi-copy transformants needed. Disadvantage over P. pastoris: - Generally lower secretion efficiency. - Grown at lower cell density. N.B. P. pastoris has strong preference for aerobic growth.
20 Case Study: megf secretion in P. pastoris vs. S. cerevisiae (Clare et al., 1991) Host S. cerevisiae Single-copy Multi-copy Secretion Level YNB Medium* YP Medium** mg/l mg/10 11 cells mg/l mg/10 11 cells < P. pastoris Single-copy copy N.D. - *YNB minimal, defined medium; YP** - complex, rich medium
21 megf secretion by multi-copy P. pastoris (Clare et al., 1991) Copy No Shake flask (mg/l) Fermenter 33.6 N.D. N.D (mg/l) Cell density = 85mg/ml (dry weight) Increase in megf levels with increased gene dosage was reduced in the fermenter c.f. shake flasks. Saturation of secretion pathway was not observed even at high gene dosage.
22 Table 5: Typical expression and purified peptide yields for P. pastoris and S. cerevisiae. Host Extracellular Intracellular Purified Peptide Secretion Yield Secretion Yield Yield (mg/l) (mg/l) (mg/l) P. pastoris N.D S. cerevisiae
23 Fusion systems for peptide expression Choice of fusion method depends on cleavage methods (chemical or enzymatic). Chemical Cleavage (CC) - CC reagents generally recognize single or paired a.a. residues (Useful for short peptides). - Removal of reagents via a dialysis/buffer exchange step. CC Reagent Cyanogen bromide N-chloro succinimide, BNPS-skatole Dilute acid Hydroxylamine Cleavage site Methionine-Xaa bond Tryptophan residues Aspartyl-prolyl (or glycine) bond Asparagine-glycine bonds at ph 9
24 Optimising cleavage site for high recovery Simultaneous cell lysis and IB fusion protein hydrolysis (without prior IB solubilisation) with 10% acid (Gavit et al., 2000). Aspartyl-prolyl bonds: acid-labile Table 6: Purification of anti-fungal peptide (Gavit et al., 2000)
25 Enzymatic Cleavage Table 7: Enzymatic methods for fusion protein cleavage (Flaschel and Friehs, 1993).
26 Thioredoxin (Trx) Da, soluble cytoplasmic protein. - structural similarity to mammalian protein disulfide isomerase (PDI) folding partner for IB-prone proteins. - can be expressed as a soluble form to 40% of total bacterial protein. Example of a fusion thioredoxin-peptide gene: factor X a cleavage site product - typical soluble yield: 10-20% of total protein (LaVallie et al., 1993; Wilkinson et al., 1995). - Main disadvantage: slow cleavage reaction time.
27 Ubiquitin (Ub) - 76 a.a. residue eukaryotic protein (with roles in protein turnover and stress response). - Ub fusion proteins have been reported in yeast (McDonnell et al., 1989; Poletti et al., 1992) and E. coli (Butt et al., 1989; Cherney et al., 1991). - Ub-specific proteases cleave fused proteins from Ub C-term. - Ub fusion partner enhances overall yields and exhibits some chaperonin-like properties.
28 Ubiquitin (Ub) fusion systems in E. coli Expression level so far: - 20 to 50% of the total cell protein in E. coli. - Peptide libraries in E. coli as Ub-fusions 60 mg/l culture (Labean et al., 1992). - Multigram peptide yields for 10 L fermentation reported: ~ 360 mg/l pure peptide obtained post-purification from Ub-fusions (Pilon et al., 1997).
29 Final Yield = 0.36 g/ L pure peptide Fig. 4: Flow diagram of the Ub fusion process for making recombinant peptides in bacteria (Pilon et al., 1997).
30 SUMO (Small Ubiquitin-like MOdifier) - ~ 100 a.a. residues, heat-stable, highly compact globular structure. - Enhances peptide expression level, improves solubility and biological activity. - Addresses problems encountered in most fusion systems (MBP, GST, Trx) where: (i) Cleavage proteases require linkers between fusion tag and peptide, leading to artificial N-terminus after cleavage. (ii)recognition sequences for proteases in linker is small, hence identical a.a. sequences in peptide may also be cleaved.
31 SUMO Proteases - highly efficient and recognises the tertiary sequence of SUMO. - cleave at the junction of fusions to release peptides.
32 SUMO Fusion Systems Fig. 5: SDS PAGE analysis showing the cleavage of 20 fusion peptides, each having a different amino acid residue following the cleavage site (LifeSensor Inc.). - Releases peptides with any desired N-term residue (except proline) gives desired N-term for enhanced bioactivity.
33 SUMO Fusion Systems SUMO proteases cannot tolerate high chaotrope concentrations. Fig. 6: SDS PAGE analysis showing SUMO protease tolerance against different chemicals (LifeSensor Inc.).
34 SUMO Fusion System Recovery Flowsheet Cell lysis Cell debris Ni- chromatography purification Elute fused peptide Fused Peptide Cleavage (with SUMO protease) Ni chromatography purification Remove fusion tag, protease Purified peptide
35 Concluding Remarks - In general, mg amounts of purified peptides achievable to date in laboratories. - P. pastoris can be favourable over E. coli for industrial production of peptides due to ease of scale up to very high cell densities, high volumetric yields and the absence of endotoxins. - Characteristics of each peptide determine the optimal host and production strategy.
36 Thank You
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