Expression of clones genes

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

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.

Pribnow box (-10)

T89 A99 T50 A65 A65 T100

70 binding site (-35)

T85 T83 G81 A61 C69 A52

E. coli

E. coli promoters, and hybrid promoters tacI and II

E. coli transcriptional termination (-independent)

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.

The pET vector system for protein production.

Control of expression by the CI repressor on the Ptrp promoter

Expression by the PBAD

promoter and its regulation

(in pBAD vectors).

Inclusion bodies in E. coli

(Scanning electron micrograph) (Thin section through the cells)

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.

Different types of translational fusion constructs.

S : signal sequence ; I : membrane integration domain ; A, B : fusion peptides, e.g. affinity tags

Examples of affinity tags

Protein production

and processing with

the pBAD/His vectors

C-terminal fusion

6 HIS-residues for affinitypurification

cleavage by enterokinase(D-D-D-D-K-*)

after processing a few extraamino acids remainat the N-terminus.

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))

Splicing of an intein

~100 amino acids ~50 amino acids

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

pMAL expression vectors malE : encodes maltose binding protein.

Properties of pMAL vectors :

ColE1 ori ; M13 ori ; Ptac ; rrnB terminators ; lacIq ; 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 fusion in XmnXmnI : exact joining of target protein at factor Xa sequenceI : exact joining of target protein at factor Xa sequence

10 Asp residues (D) separate the two fusion moieties10 Asp residues (D) separate the two fusion moieties

insertion in insertion in EcoEcoRI is identical (in reading frame) as in RI is identical (in reading frame) as in gt11 (gt11 (lacZlacZ))

Cleavage site ofCleavage site of factor Xafactor Xa I - E - G - R I - E - G - R **

XmnXmnI I nnn nnn nnnn nnn nGA AGA Ann nnnn nnT TCT TCn nnn n nnn ATC-GAG-GATC-GAG-GGA-AGA-AGG-ATGG-ATT-TCT-TCA-A-GAA-TTCGAA-TTC--

EcoEcoRI RI

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)

Processing of expressionproducts from fusionconstructs in pMAL vectors.

(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 bystreptavidin-alkaline phosphatase.

Elution of fusion protein with5 mM biotin.(hence : no denaturation required)