Profinity eXact Expression Vector Kits and Cloning Products

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Overview

Kits and competent cells for cloning and expression of Profinity eXact fusion-tagged recombinant proteins.
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Description

Description

The pPAL7 vectors are derived from a T7-based expression plasmid and utilize the T7 lac promoter and terminator, and a T7 RNA polymerase expression host for inducible protein production. The plasmids confer ampicillin resistance, constitutively express the lacI repressor for tight control of target gene transcription, and have a pMB1-derived ColE1 origin of replication. Gene targets cloned into Profinity eXact pPAL7 vectors express the prodomain of the subtilisin protease as their fusion partner or tag.

Two pPAL7 expression vector kits are available for the Profinity eXact fusion-tag system – the pPAL7 RIC-ready expression vector kit and the pPAL7 supercoiled expression vector kit. Both contain a 5901-bp pPAL7 vector, but the former contains a versatile predigested form of plasmid for restriction enzyme-free cloning of any target gene, regardless of internal restriction sites, and the latter, a supercoiled plasmid.

Precise fusions in which the resultant purified product does not contain any additional amino acids at the N-terminus may be constructed using the HindIII restriction site and an appropriate restriction site in the multiple cloning site (MCS), or by cloning with the prepared restriction-independent cloning (RIC) vector. Use of the linearized, RIC-ready vector facilitates high-throughput cloning of DNA sequences, regardless of the restriction sites that may be present within a target gene.

Profinity eXact pPAL7 RIC-Ready Expression Vector Kit
The pPAL7 restriction-independent cloning (RIC)-ready expression vector is designed to allow higher throughput and versatility for the cloning of target DNA sequences. The vector is prepared by double digestion of the supercoiled plasmid with SapI and EcoRI, and is then dephosphorylated to reduce postligation background transformants. The target DNA is amplified by PCR using phosphorylated primers containing specific residues at the 5' end that, upon treatment with T4 DNA polymerase, create 5' overhangs complementary to the RIC-ready vector.

Profinity eXact pPAL7 Supercoiled Expression Vector Kit
The pPAL7 supercoiled plasmid expression vector is used for routine cloning of target DNA sequences using a conventional restriction digest cloning strategy. Both the pPAL vector and PCR product are digested with appropriate enzymes found in the multicloning site and ligated.

BL21 (DE3) Chemi-Competent Expression Cells
Chemi-competent cells are also available for cloning and expression of Profinity eXact fusion-tagged proteins. BL21 (DE3) E. coli cells* are the preferred host for T7 vector based protein expression. The cells are λDE3 lysogens with the T7 RNA polymerase gene under the control of the lacUV5 promoter. Induction with IPTG allows production of T7 RNA polymerase, which then directs the expression of the target gene located downstream of the T7 lac promoter in the expression vector. The strain is deficient in ompT and lon proteases, which provides improved recombinant protein stability.

* Genotype: E. coli B F– dcm ompT hsdS(rB –, mB –) gal λ(DE3).

Profinity eXact Cloning and Expression Starter Kit
The Profinity eXact cloning and expression starter kit includes everything necessary to clone and express target genes using the Profinity eXact system technology. This comprehensive kit includes both Profinity eXact pPAL supercoiled and RIC-ready expression vectors, chemi-competent cloning cells, BL21 (DE3) chemi-competent expression cells, and SOC growth medium.

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1563001
Includes 25 μl of 20 ng/μl pPAL7 restriction-independent cloning (RIC)-ready expression vector; sufficient for 20 reactions
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1563002
Includes 100 μl of 100 ng/μl pPAL7 supercoiled plasmid expression vector; sufficient for 20 reactions
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1563003
Pkg of 10 vials, 0.05 ml BL21 (DE3) E. coli cells, also includes pUC 19 control plasmid, 10 ml vial of SOC growth media
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1563000
Includes 25 µl of 20 ng/µl pPAL RIC-ready and 100 µl of 100 ng/µl pPAL supercoiled expression vectors, chemi-competent cells, SOC growth media; sufficient for 20 reactions

Documents

Documents

Number Description Options
5652
Profinity eXact Fusion-Tag System Performs On-Column Cleavage and Yields Pure Native Protein From Lysate in Less Than an Hour, Rev A
5656
Purification of Tag-Free Recombinant Proteins Using the Profinity eXact Fusion-Tag System, Rev B
5668
Profinity eXact Fusion-Tag System Brochure, Rev A
5655
Profinity eXact™ Resin Product Information Sheet, Rev A
5646
Profinity eXact™ Cloning and Expression Kits, PIS, Rev B
5811
Profinity eXact Fusion-Tag System FAQs, Rev A
5867
The pPAL7 Vector Map and Plasmid Sequence Data Sheet, Rev B
5898
Using the Profinity eXact Fusion-Tag System: Strategies for Success, Rev A
10011161
BL21(DE3) Chemi-Competent Expression Cells Manual, Rev A
10011260
Profinity eXact Protein Purification System Manual, Rev A
5772
Periplasmic Expression and Purification of Recombinant Proteins from E.coli Using the Profinity eXact Fusion-Tag System, Rev A
6590
Chromatography Column Performance and Data Analysis Guide, Rev A
5928
Application of the Profinity eXact Fusion-Tag System to Eukaryotic Expression Systems Tech Note, Rev B