PLASMID REFERENCES AND NOTES

Bacterial Expression Vectors#1

pJL3 differs from earlier pJL vectors, including pJL2, by the removal of unnecessary restriction sites introduced during construction.  Two 5' restriction sites, Xba1 and Nco1, can be used for insertion of the desired cDNA coding sequence, with a 3' Hind3 site.  

pINIIIompA3 (1) attaches the outer membrane protein A signal peptide (21 amino acids) to the N-terminus of the cloned protein.  This directs the nascent polypeptide to the inner membrane of the bacterium, where it is either secreted into the periplasm or becomes anchored to the inner membrane.  Signal peptidase on the periplasmic side of the membrane removes the signal peptide during export, but with some membrane-bound proteins the signal peptide is not removed (2).  The vector uses the lpp-lac promoter and thus is IPTG-inducible.  Translation is highly efficient due to the use of the ompA translation initiation region; three cloning sites at end of signal peptide can be used for cDNA insertion.  The cDNA coding sequence must be in-frame with the signal peptide sequence.  See note below regarding sequence errors.

A high-copy version of pINIIIompA3 in which the pBR322 origin of replication is replaced by the origin from the pUC series of plasmids (1).  Expression of some proteins with this vector can be unsuccessful due to excessive protein production and resulting toxicity.  See note below regarding sequence errors.

P450 2E1 Expression Vectors#2

Full-length rabbit P450 2E1 cDNA (1,2) in pJL2 (3).  Expression is induced by addition of IPTG to 1 mM.
Good expression after 4 hr induction at 37° (>20 nmol/L)

Full-length human P450 2E1 in pJL2 vector (1).  Second codon (TCT, Ser) changed to GCT (Ala) to accommodate cloning into Nco1 site of vector.  Expression is induced by addition of IPTG to 1 mM.  4-Methylpyrazole, a P450 2E1 inhibitor, can be included in cultures (5 uM) and preparation buffers (50 uM) to stabilize P450 2E1. 
Poor expression even after 2-3 day induction at room temp (<1 nmol/L)  

Full-length human P450 2E1 (1) in pINIIIompA3 vector (2,3).  Signal peptide is not removed, leaving the 21 amino acid signal peptide along with 3 intervening amino acids (Gly-Ile-Thr) attached to the N-terminus of the enzyme. Second amino acid of P450 2E1 sequence changed from Ser to Ala to accommodate cloning.  Expression is induced by addition of IPTG to 1 mM.  4-Methylpyrazole, a P450 2E1 inhibitor, can be included in cultures (5 uM) and preparation buffers (50 uM) to stabilize P450 2E1. 
Good expression after 2-3 day induction at room temp (10-20 nmol/L)
See note below regarding sequence errors.

A high-copy version of pIN3/2E1_human.  Expression is induced by addition of IPTG to 1 mM.  4-Methylpyrazole, a P450 2E1 inhibitor, can be included in cultures (5 uM) and preparation buffers (50 uM) to stabilize P450 2E1. 
Good expression after 2-3 day induction at room temp (15-30 nmol/L)
See note below regarding sequence errors.

P450 Reductase Expression Vectors#3

Full-length rat cytochrome P450 reductase cDNA in pINIIIompA3 (1,2).  Identical to pOR262 in (2).  OmpA signal peptide is efficiently cleaved from protein, leaving 8 amino acids attached to N-terminus (created during cloning).  Expression induced with 1 mM IPTG, 4 hrs to overnight at 37°.  High expression (>50 nmol/L)
See note below regarding sequence errors.

Identical to pIN3/OR, but with high copy number origin of replication.
See note below regarding sequence errors.  

P450 2E1/Reductase Coexpression Vectors

Full-length human P450 2E1 cDNA (1) upstream of rat cytochrome P450 reductase cDNA (2,3) in pJL2 vector (1).  Second codon of P450 (TCT, Ser) changed to GCT (Ala) to accommodate cloning.  23 base-pairs separate P450 termination codon from start codon of reductase sequence, and includes a ribosome binding site.  Note that the reductase cDNA is preceded by the ompA signal peptide to enhance membrane incorporation; the signal peptide is efficiently removed from the reductase in vivo by signal peptidase, leaving 8 intervening amino acids incorporated during cloning.  Expression is induced by 1 mM IPTG for ~60 hr at room temperature with slow shaking.  P450 expression is poor (<1 nmol/L); reductase expression is good (>20 nmol/L).  4-Methylpyrazole, a P450 2E1 inhibitor, can be included in cultures (5 uM) and preparation buffers (50 uM) to stabilize P450 2E1. 

Full-length human P450 2E1 cDNA (1) upstream of rat cytochrome P450 reductase cDNA (2,3) in pINIIIompA3 vector (4).  Second codon of P450 (TCT, Ser) changed to GCT (Ala) to accommodate cloning.  23 base-pairs separate P450 termination codon from start codon of reductase sequence, and includes a ribosome binding site.  Note that both the P450 and reductase cDNAs are preceded by the ompA signal peptide.  This peptide is efficiently cleaved from the reductase protein leaving 8 intervening amino acids incorporated during cloning, but remains attached to P450 2E1 along with 3 intervening amino acids.  Expression is induced by 1 mM IPTG for ~60 hr at room temperature with slow shaking.  P450 expression is low (~20 nmol/L); reductase expression is good (>50 nmol/L).  4-Methylpyrazole, a P450 2E1 inhibitor, can be included in cultures (5 uM) and preparation buffers (50 uM) to stabilize P450 2E1. 
Used in the recombinant human CYP2E1 Ames mutagenicity test (pIN3ER) (5,6).
See note below regarding sequence errors.

Identical to pIN3/2E1/OR, but with the pUC high copy origin of replication to increase protein expression levels.  Expression is 60-70% higher than that obtained with pIN3 construct (1).  
See note below regarding sequence errors.

P450 2E1/Reductase/Cytochrome b₅ Coexpression Vector#3

Full-length human cytochrome b₅ (1) placed upstream of the human P450 2E1 and rat cytochrome P450 reductase cDNAs (2,3,4) in pINIIIompA3 vector (5).  Thirty nucleotides separate the b₅ and P450 cDNAs, including a ribosome binding site.  Second codon of P450 (TCT, Ser) changed to GCT (Ala) to accommodate cloning.  23 base-pairs separate P450 termination codon from start codon of reductase sequence, and includes a ribosome binding site.  Note that both the P450 and reductase cDNAs are preceded by the ompA signal peptide.  This peptide is efficiently cleaved from the reductase protein leaving 8 intervening amino acids incorporated during cloning, but remains attached to P450 2E1 along with 3 intervening amino acids.  Expression is induced by 1 mM IPTG for ~60 hr at room temperature with slow shaking.  P450 and b₅ expression is low (~20 nmol/L); reductase expression is good (>50 nmol/L).  4-Methylpyrazole, a P450 2E1 inhibitor, can be included in cultures (5 uM) and preparation buffers (50 uM) to stabilize P450 2E1. 
Used in the recombinant human CYP2E1 Ames mutagenicity test (pIN3b₅ER) (6).
See note below regarding sequence errors.

Squalene Monooxygenase Expression Vector#3

Human squalene monooxygenase (SE) cDNA in pTYB4 expression vector (New England Biolabs) (1).  The first 110 amino acids of SE, which contain the membrane binding domain, have been removed to increase expression.  Thus, Ile110 is replaced by Met (start codon) and Gly111 is replaced by Ala.  This does not appear to affect activity with purified preparations.  Expression uses the IPTG-inducible T7 promoter, and requires the use of E. coli strains containing T7 polymerase (i.e., ER2566 [New England Biolabs] or BL21[DE3], [Stratagene]).  Expression is induced with 1 mM IPTG, overnight at 30°.  SE is expressed as a fusion to the self-cleaving intein sequence, followed by a chitin-binding domain (CBD).  Cell lysates are passed over a chitin column (New England Biolabs) and, following washing, the column is incubated overnight at 4° in 30 mM 2-mercaptoethanol to promote cleavage.  SE is then eluted with wash buffer. 

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Comments to Todd D. Porter, Pharmaceutical Sciences, University of Kentucky College of Pharmacy, Lexington, KY 40536-0082.  
Phone 859 257-1137; FAX 859 257-7564
Last Modified: December 02, 2001
Copyright © 2000, University of Kentucky Chandler Medical Center