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Expression of clones genes a.o. Primrose & Twyman, 7th edition, pp.81-93 Primrose, Twyman & Old, 6th edition, pp.70-83.

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Presentation on theme: "Expression of clones genes a.o. Primrose & Twyman, 7th edition, pp.81-93 Primrose, Twyman & Old, 6th edition, pp.70-83."— Presentation transcript:

1 Expression of clones genes a.o. Primrose & Twyman, 7th edition, pp.81-93 Primrose, Twyman & Old, 6th edition, pp.70-83

2 IPTG-induced expression of RP4 proteins : (A) DNA primase (118 and 80 kDa proteins) ; (B) 16.5 and 8.6 kDA proteins. Host strain : E. coli HB101. Analysis by SDS-polyacrylamide gel electrophoresis. (A) insert in direct (c, d) or opposite (e,f) orientation ; induced (c, e) or non-induced (d,f) with IPTG. (a) reference proteins ; (b) purified RP4 DNA primase (B) insert in direct (b, c) or opposite (d,e) orientation ; induced (b, d) or non-induced (c,e) with IPTG. (f) reference proteins ; (a) purified 16.5 kDa protein.

3 Pribnow box (-10) T 89 A 99 T 50 A 65 A 65 T 100  70 binding site (-35) T 85 T 83 G 81 A 61 C 69 A 52 E. coli E. coli promoters, and hybrid promoters tacI and II E. coli transcriptional termination (  -independent)

4 Codeword usage in strongly and weakly expressed E. coli genes. "strong" :24 genes (5253 triplets), a.o. 12 of ribosomal proteins, 7 of outer membrane proteins, and some for RNA polymerase subunits and translation initiation and termination factors. "weak" : 18 genes (5231 triplets), a.o. for different repressor proteins, lac-permease, etc.

5 The pET vector system for protein production.

6 Control of expression by the CI repressor on the P trp promoter

7 Expression by the P BAD promoter and its regulation (in pBAD vectors).

8 Inclusion bodies in E. coli (Scanning electron micrograph)(Thin section through the cells)

9 Constitutive expression of the cat gene, and fusions by random cloning into the ScaI site (lane 2)(lanes 1, 6, 7, 9-15) Lanes 4 and 5 are plasmid-free cells ; lanes 3 and 8 are protein size markers.

10 Different types of translational fusion constructs. S : signal sequence ; I : membrane integration domain ; A, B : fusion peptides, e.g. affinity tags

11 Examples of affinity tags

12 Protein production and processing with the pBAD/His vectors C-terminal fusion 6 HIS-residues for affinity purification cleavage by enterokinase (D-D-D-D-K-*) after processing a few extra amino acids remain at the N-terminus.

13 C-terminal fusion at biotin carboxylase Binding onto streptavidin column for purification. Processing with factor Xa protease (I-E-G-R-* of I-D-G-R-* (not followed by P or R) )

14 Splicing of an intein ~100 amino acids ~50 amino acids

15 Processing of N-terminal fusions at an intein, immobilized onto chitin columns

16 pMAL expression vectors malE : encodes maltose binding protein.

17 Properties of pMAL vectors : ColE1 ori ; M13 ori ; P tac ; rrnB terminators ; lacI q ; bla Fusion construct : malE - DDDDDDDDDD - IEGR - MCS(polylinker) - lacZ  pMAL-c2 : malE signal peptide sequence deleted pMAL-p2 : malE signal peptide sequence : secretion to periplasm  fusion in XmnI : exact joining of target protein at factor Xa sequence  10 Asp residues (D) separate the two fusion moieties  insertion in EcoRI is identical (in reading frame) as in gt11 (lacZ) Cleavage site of factor Xa I - E - G - R * XmnI nnn nnn nGA Ann nnT TCn nnn ATC-GAG-GGA-AGG-ATT-TCA-GAA-TTC- EcoRI EcoRI

18 Multiple cloning site in (one of) the pMAL vectors. Other possibility (in some vectors) Pro-Gly-Ala-Ala-His-Tyr : cleavage by Genenase I = engineered form of subtilisin : cleaves beyond His-Tyr (depending on the next residue) (cleaves His-Tyr-Glu and His-Tyr-Asp slowly, doesn't cleave His-Tyr-Pro or His-Tyr-Ile)

19 Processing of expression products from fusion constructs in pMAL vectors.

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21 (Commercial) Pinpoint vectors two promoters : T7 and tac factor Xa cleavage site N-terminal biotinylation (tag) polylinker in vitro RNA probes by SP6 promoter secretion signal (some vectors) Biotinylation at a Lys residue (a single biotin) in E. coli by the biotin ligase holoenzyme. Accessible to avidin or streptavidin ; tag for detection and purification. nb. E. coli produces a single endogenous biotinylated protein that, in its native conformation, does not bind to avidin : => highly specific for the recombinant fusion protein Detection by streptavidin-alkaline phosphatase. Elution of fusion protein with 5 mM biotin. (hence : no denaturation required)

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