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EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
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EXPRESSION SYSTEMS
Dr. Mario Lebendiker
mariol@cc.huji.ac.il
Tel: 972-2-6586920 
 
Bacterial Exression Systems

GST
poly His
Maltose
Calmodulin
Intein
Biotinylated
Two Step: poly His and Strep-Tag
Solubility enhancement tags (SETs)
SUMO
Cold Expression
Single Protein Production
Profinity eXact™

Corynebacterium
Pseudomonas fluorescens
ESETEC Secretion System
Brevibacillus Expression System
Cascade

Drosophila Expression System

Mammalian

Insect

Leishmania

Yeast


Bryophytes (Mosses)
In-Vitro Translation
 

Bacterial Expression Systems 

           GST-Fusion Proteins
           PHARMACIA-GST fusion protein Handbook (pdf) 

        poly His-Tagged Fusion Proteins
               
                CLONTECH:  BD HAT Protein Expression and Purification System User Manual  (pdf) 
           NOVAGEN:   pET purification manual   (pdf)   (pdf-2)    
               
QIAGEN: Ni-NTA purification manual (pdf)
 

        Maltose Binding Protein
           NEW ENGLAND BIOLAB: pMAL protein Fusion and Purification System (pdf) 

        Calmodulin Binding Peptide 

             STRATAGENE: CBP Calmodulin-Binding Peptide Affinity Tag System  (pdf-I)    (pdf-II)     (pdf-III)

        Intein Mediated Purification 

             NEW ENGLAND BIOLAB:  The IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein        
                    purification system which utilizes the inducible self-cleavage activity of protein splicing elements (termed inteins) to separate the target protein 
                    from the affinity tag. Each intein tag contains a chitin binding domain (CBD) for the affinity purification of the fusion protein on a chitin resin. 
                    Induction of on-column cleavage, using thiol reagents such as dithiothreitol (DTT), releases the target protein from the intein tag. 

                    Able to produce target protein without vector–derived amino acids
 (pdf)
                    INTEIN-TM System (pdf)

                    INTEIN-TWIN System (pdf)

            Biotinylated Fusion Protein

                PROMEGA    PinPointTM Xa Protein Purification System  (pdf)    

        Two Step: poly His and Strep-Tag
              IBA:  Expression and purification of proteins using Strep-tag and/or 6xHistidine-tag. A comprehensive manual. The Strep-tag II is a short
                    peptide (8 amino acids, WSHPQFEK), which binds with high selectivity to Strep-Tactin, an engineered streptavidin. Elution of purified
                    recombinant  protein is performed by desthiobiotin.
 (pdf)

              IBA  Tools for protein expression & purification. Strep-tag® technology and 6xHis-tag & Ni-NTA technology: Double tag protein expression
                    and purification
 (pdf) 

              NOVAGEN   Strep•Tactin® Purification Kits. Strep•Tactin protein is a streptavidin derivative developed for optimal Strep•Tag II binding. The
                    binding affinity of Strep•Tag II for Strep•Tactin is approximately 100 times higher than for streptavidin. The purified target protein is
                    competitively eluted with 2.5 mM desthiobiotin, an analog of biotin that reversibly binds Strep•Tactin. Multi-system: E. coli, baculovirus,
                    vectors   incorporate the Strep•Tag® II fusion tag. Enterokinase (Ek) or HRV 3C (3C) cleavage site. Vectors for secretion of fusion proteins
                    from insect cells, or both insect and mammalian cells, and export to the periplasm in E. coli cells
     (pdf-I)    (pdf-II)

              QIAGEN:   Two-Step Affinity Purification System Handbook 
                                 For expressing, purifying, and detecting proteins carrying a 6xHis and Strep-tag II
 (pdf)

            Solubility enhancement tags (SETs)

            STRATAGENE:    VariFlex™ bacterial protein expression system. Include three different solubility enhancement tags (SETs) which are
                designed to increase protein solubility, the streptavidin binding peptide (SBP) purification tag, and a tag that allows for rapid soluble protein
                quantification (Q-tag)
   (pdf)
   
           SUMO

               INVITROGEN:   The Champion™ pET SUMO Protein Expression System utilizes a small ubiquitinlike modifier (SUMO) to allow expression,
                    purification, and generation of native proteins in E. coli. SUMO fusions may increase the expression of recombinant proteins and enhance the
                   solubility of partially insoluble proteins. In addition, the tertiary structure of the SUMO protein is specifically recognized and cleaved by a
                   ubiquitin-like protein-processing enzyme, SUMO Protease resulting in the production of native protein.
 (pdf)

               LIFESENSORS  The SUMOpro Protein and Peptide Expression Kit maximizes the yield of soluble, untagged, functional proteins with desired
                    N-termini. The system use an  highly active and specific SUMO protease 1 that recognizes the natural tertiary structure of the SUMO (Smt3) tag,
                    generating the desired N-terminus and never cleaving within your protein of interest. By linking a 6XHis epitope to both the SUMO fusion tag
                    and SUMO Protease 1, the SUMOpro system allows efficient and rapid purification of untagged protein using Ni-NTA chromatography.   (pdf)    

               LIFESENSORS  The SUMOpro-3 Protein and Peptide Expression Kit maximizes the yield of soluble, untagged, functional proteins with desired
                    N-termini.  The system uses an active and specific SUMO Protease 2 that recognizes the natural tertiary structure of the SUMO (HuSUMO-3) tag,
                    generating the desired N-terminus and never cleaving within your protein of interest.  By linking a 6XHis epitope to both the SUMO-3 fusion tag
                    and SUMO Protease 2, the SUMOpro-3 system allows efficient and rapid purification of untagged protein using Ni-NTA chromatography.
                    The SUMOpro-3 kit is recommended for expression and purification of proteins that contain many hydrophobic residues at or around the
                    N-terminus.   (pdf)

               LIFESENSORS  The SUMOstar Protein and Peptide Expression Kit offers an excellent system to maximize production of untagged recombinant
                    proteins with a desired N-terminus. The SUMOstar E. coli system provides excellent protein yield for anyone who wishes to ultimately express
                    a protein in a eukaryotic host, but wishes to first test the effects of SUMOstar fusion in a more cost-effective E. coli system.
                    The system uses anactive and specific SUMOstar Protease 1 instead recognizes the tertiary structure of the SUMOstar tag, generating the
                    desired N-terminus and never cleaving within your protein of interest. By linking a 6XHis epitope to both the SUMOstar fusion tag and
                    SUMOstar Protease 1, the SUMOstar system allows efficient and rapid purification of untagged protein using Ni-NTA chromatography. (pdf)

               LIFESENSORS  SUMO Gene Fusion Technology - New Methods for Enhancing Protein Expression and Purification in Prokaryotes   (pdf) 

               LIFESENSORS  SUMO Gene Fusion Technology - New Methods for Rapid Recombinant Peptide Expression and Purification   (pdf)


      Cold Expression

                TAKARA  pCold TF DNA Vector is a fusion cold shock expression vector that expresses Trigger Factor (TF) chaperone as a soluble tag
                    (48 kDa) which facilitates co-translational folding of newly expressed polypeptides. The vector has a His-Tag sequence, and a multicloning site
                    (MCS). A lac operator is inserted downstream of  the cspA promoter to ensure strict regulation of expression. Additionally, recognition sites
                    for HRV 3C Protease, Thrombin, and Factor Xa are located between TF-Tag and the Multiple Cloning Site (MCS) and function to facilitate
                    tag removal from the expressed fusion protein. Most E. coli strains can serve as expression hosts. The pCold TF DNA Vector provides cold
                    shock technology for high yield protein expression combined with Trigger Factor (chaperone) expression to facilitate correct protein folding,
                    thus enabling efficient soluble protein production for otherwise intractable target proteins.   (pdf-I)   Chaperone Plasmid Set / Chaperone
                    Competent Cells
(pdf-II)    

       Single Protein Production

                 TAKARA   Single Protein Production system (SPP systemTM). This system utilizes an E. coli protein MazF, a sequence-specific
                    endoribonuclease which cleaves single strand RNAs at ACA sequences. In this system, the transcript of interest which should not contain
                    any ACA sequences (i.e. ACA-less), and MazF are co-expressed in a host Escherichia coli. Therefore the MazF does not cleave the
                    transcript of interest, but cleave the ones derived from the host proteins or others at ACA sequences. So, only the transcript of interest is
                    dominantly translated and only the protein of interest is dominantly expressed, with the SPP. In order to construct SPP system, it is first
                    necessary to prepare an ACA-less gene of interest by chemical synthesis or site-directed mutagenesis technology, etc.  (pdf)

           Profinity eXact™

                  BIO-RAD   Profinity eXact fusion-tag system is an affinity tag-based protein purification system that  utilizes a modified form of the subtilisin
                     protease, which is immobilized onto a chromatographic support and used to generate pure, tag-free target protein in a single step.
                     The tag in this system is the prodomain (prosignal sequence) of the subtilisin protease, a 75-amino acid sequence that is fused to the
                     N-terminus of a target protein of interest. The mature and prodomain subtilisin protease sequences have been co-engineered to produce a
                     highly specific, high-affinity interaction between the binding partners. Application of elution buffer triggers subtilisin’s processing activity,
                     which quickly and precisely cleaves the tag from the fusion protein and releases the purified target protein. At the end of the purification
                     process, the tag remains tightly bound to the resin and contains only its native amino acid sequence. The structure and activity of the native
                     protein is maintained, and the need for additional materials, such as cleavage enzymes and purification resin for postcleavage enzyme removal,
                     is eliminated.  (pdf-I)  (pdf-II)   (pdf-III)  Profinity eXact purification resin  (pdf-IV)   (pdf-V)

                Corynebacterium
       
                    CORYNEX    Corynex Expression System. Simplified expression process.Gram-positive, fast growing soil bacterium. Nonpathogenic. Secretes
                    active proteins. Engineered to eliminate host protein secretion. Fewer purification steps. Fewer unwanted byproducts (proteases).
                    High efficiency / no capacity constraints. Wide range of target proteins capabilities: including complicated r-Proteins.  (pdf)

                 Pseudomonas fluorescens

                    PFENEX  Pfēnex Expression Technology™ is a robust Pseudomonas fluorescens-based platform uniquely employing a high-throughput
                    methodology for identification of optimized strains for therapeutic protein and vaccine production. Secretion of proteins to the periplasm of
                    the P. fluorescens cell, while simultaneously over-expressing folding modulators, enables the proper formation of disulfide bonds. Subsequently
                    in order to recover the protein, the use of periplasmic release methods selectively permeabilizes the outer membrane of the cell, while keeping the
                    inner membrane intact resulting in a much purer intermediate. Periplasmic extracts obtained with  Pfēnex Expression Technology are of much
                    higher purity than standard, whole cell bacterial lysates thus simplifying downstream purification processes with increased overall yields. A large
                    collection of protease knockout host strains helps to ensure that the expressed protein is not subject to proteolytic clipping or degradation.
                    P. fluorescens
will not glycosylate proteins, thus avoiding the costly problem of extensive heterogeneity frequently encountered in yeast expression
                    systems.  (pdf-I)  (pdf-II)  (pdf-III)  (pdf-IV)

                   ESETEC Secretion System

                    WACKER BIOTECH  ESETEC® technology provides an innovative and highly efficient E. coli expression system, which enables secretion of native    
                    recombinant protein products into the fermentation broth. This simplifies primary recovery and purification processes.

                          The system is based on a two-step export mechanism:

                            - First, the target product is transported across the cyto- plasmatic membrane into the periplasmatic space via the sec-pathway. During this step the
                    signal peptide is cleaved off, releasing the native product.

                            - The second step is mediated by a unique feature of the proprietary WACKER Secretion Strain. The correctly folded product is secreted from the                                         periplasmatic space across the outer membrane into the culture broth.
                    Yields as high as 7 g/l have already been obtained. E. coli K12 derivative, biosafety level , genetically well characterized; stable in commercial-scale fermentation                             Different genetic variants available, e.g. protease deletion mutants.                           
                           Additional Helper Elements of the ESETEC® System:  Cytoplasmic chaperones.  Components of the secretion apparatus. Periplasmic chaperones.
                            Disulfide bridge formation factors.    (pdf-I)   (pdf-II) 

                    Case Study: Production of a HuCAL® Fab Fragment using the WACKER Secretion System   (pdf-III)

             
                    Brevibacillus Expression System
                
TAKARA   Brevibacillus (Bacillus brevis) Expression System is an excellent and high-efficient protein production system. This bacterium, a
                        Gram-positive microbe, is characterized in particular by its ability to secrete/produce large amount of proteins. This characteristic has been
                        successfully used in the secretory production of a large number of heterologous proteins. Recent studies have shown that this bacterium is
                        excellent not only in secretory productions but also in intracellular productions. Proteins that insolubilize and precipitate when produced by
                        E. coli
may be recovered in a soluble state when produced by this bacterium. This system is recommended for the production of functional
                        proteins that are naturally produced intracellularly but  become insolubilized and cannot undergo in vitro refolding when produced by E. coli.
                        This system has the following features:
                                efficient intracellular production of soluble heterologous proteins
                                easy to culture and sterilize
                                amenable to genetic manipulations
                                a safe host
                        With high transformation efficiency by electroporation, this host is amenable to genetic manipulation. Using a shuttle vector between this bacterium
                        and E. coli, expression vectors can be constructed in E. coli. A vector with a His-tag inserted to the N-terminus is also available. The target
                        protein can be easily purified using a histidine tagged protein purification column. The His-Tag can be removed by exposing the purified protein to                                     enterokinase treatment.    (pdf)


                     CASCADE

                   BIOMEDAL  CASCADETM  is a bacterial protein expression system that provides tightly regulated, high-level expression. The system makes use
                    of linked regulatory circuits to amplify gene expression levels when induced, maintaining low basal expression levels under non-inducing conditions.
                    Tight control of gene expression. Stable phenotypes by using mini-Tn5 mediated integration. Active at low temperature (16ºC) and low sensitivity to
                    media formulation. Availability of vectors with protein fusion tags for purification using low cost affinity supports. Expression of heteromultimeric
                    proteins.  (pdf-I)   (pdf-II)   (pdf-III)

Drosophila Expression System

             INVITROGEN :  Drosophila Expression System  (pdf)


Insect Expression System

                LIFESENSORS   Insect Intracellular SUMOstar Expression System offers the combined advantages of enhanced expression with SUMO
                    fusion technolgy and the post-translational modification potential of Insect cells.  Fusion to SUMOstar allows the highest level of expression,
                    and specific cleavage to yield native protein with desired N-termini. SUMOstar enhances expression and increases solubility in insect cells. (pdf)  

                LIFESENSORS  Insect Secretory SUMOstar Expression Kit. A specific SUMOstar protease was engineered to specifically recognize and cleave
                    the SUMOstar tag in vitro, liberating the protein-of-interest with a desired N-terminal amino acid (endogenous SUMO proteases do not cleave
                    the SUMOstar fusion construct). Fusion proteins are targeted to the secretory pathway for folding and secretion.
                    SUMOstar fusion enhances the expression level and solubility of fusion proteins in eukaryotic expression system.
                    The SUMOstar tag protects the expression host from the cytotoxic effects of many protein fusion partners.
                    The insect expression system offers an alternative to yeast expression systems, and often produces more functional protein through high-fidelity
                    post-translational modifications and chaperone-mediated protein folding.
                    Insect expression provides a cost effective eukaryotic alternative to mammalian expression systems for functional protein production. (pdf)  
           
                NOVAGEN  InsectDirect™ Protein Expression & Purification System provides rapid, plasmid-mediated expression to generate small to
                    moderate amounts of recombinant protein without creating recombinant baculovirus. This system is ideal for high throughput (HT) expression
                    screening in insect cells. BacMagic™ DNA provides faster baculovirus production by eliminating the time-consuming and labor-intensive
                    plaque purification steps.   (pdf)   (pdf-II)  

Leishmania   

                 JENA  BIOSCIENCE  LEXSY  Leishmania tarentolae Expression System  (pdf)  (pdf-II)    (pdf-article)


Mammalian Expression System

                INVITROGEN :  Xpressª System Protein Expression pEBVHis. A Manual of Methods for Expression of Polyhistidine - Containing Recombinant
                    Proteins using the pEBVHis Vector System in Mammalian Cells
 (pdf)
                  
                LIFESENSORS  Mammalian Intracellular SUMOstar Expression Kit.  SUMOstar offers the same solubility and expression enhancing benefits as
                    the original wild-type SUMO tag; however, the tag is not cleaved by endogenous eukaryotic SUMO proteases. A specific SUMOstar protease
                    was engineered to specifically recognize and cleave the SUMOstar tag in vitro, liberating the protein-of-interest with a desired N-terminal amino acid.
                    The SUMOstar Mammalian Cell Expression Kit is designed for expression of proteins in the cytosol primarily for, for example cell biological
                    or protein-protein interaction studies.  (pdf)  
       
                LIFESENSORS   Mammalian Secretory SUMOstar Expression Kit.  The secretory kit is designed for expression into the medium for production
                    of biologically relevant proteins.  The kit can also be used for expression of membrane proteins. Fusion proteins are targeted to the secretory
                    pathway for folding and secretion.
                    SUMOstar fusion enhances the expression level and solubility of fusion proteins in eukaryotic expression system.
                    The SUMOstar tag protects the expression host from the cytotoxic effects of many protein fusion partners.
                    SUMOstar Protease 1 is specifically engineered to recognize and cleave the SUMOstar fusion tag to release the protein-of-interest with a
                    desired N-terminus.  (pdf)  

Yeast Expression System


               LIFESENSORS   Saccharomyces Secretory SUMOstar Expression Kit.  SUMOstar offers the same solubility and expression enhancing benefits
                    as the original wild-type SUMO tag; however, the tag is not cleaved by endogenous eukaryotic SUMO proteases. A specific SUMOstar
                    protease was subsequently engineered to specifically recognize and cleave the SUMOstar tag in vitro, liberating the protein-of-interest with a
                    desired N-terminal amino acid (except Proline)  (pdf) 

                LIFESENSORS  Pichia Secretory SUMOpro-3 Expression Kit offers the combined advantages of enhanced protein expression with SUMO
                    fusion technology and the high-level production capabilities and scale-up potential of Pichia pastoris. Through the fusion of Human SUMO3 to
                    proteins, drastically enhanced expression has been demonstrated. Fusion partners are not only produced in greater quantity but more easily
                    purified using common affinity chromatography tags attached to the N-terminus of Human SUMO3. The alpha mating factor secretion signal
                    is incorporated into the tag to target the fusion protein to the secretory pathway. Endogenous eukaryotic SUMO proteases (localized to the
                    cytosol) are thus avoided, and the fusion protein is efficiently secreted into the extracellular medium. The highly specific SUMO protease 2
                    efficiently and specifically cleaves off the fusion tag in vitro, generating a native N-terminus on the protein of interest.  (pdf)

                 NEW ENGLAND BIOLAB:   K. lactis Expression Kit provides an easy method for expressing a gene of interest in the yeast Kluyveromyces
                     lactis. Proteins may be produced intracellularly or be secreted and have access to eukaryotic protein folding and glycosylation machinery that
                    E. coli cells do not possess, making it an important alternative to bacterial expression systems. K. lactis has been used to produce proteins at
                    industrial scale in the food industry for over a decade due to its ability to rapidly achieve high culture densities and abundantly produce
                    recombinant proteins
  (pdf)   (pdf-I)    (pdf-II)

Bryophytes (Mosses)    

                GREENOVATION    The Bryotechnology uses the small moss Physcomitrella patens as production system for the recombined or genetic
                    production of proteins. The production of proteins in mosses has the advantage that it is simple, reliable and cost efficient. Moss is the only
                    known plant system which shows a high frequency of homologous recombination. This attribute allows for targeted gene insertion as well as
                    for targeted gene knockouts. The homologous recombination of moss is a highly attractive tool for production strain design and is used to
                    knock-out plant-specific glycosylation enzymes. 
                    This permits the removal of plant-specific glycosylation patterns and retaining glycosylation patterns that are highly similar to those found in
                    human cells. The moss protonema is grown under photoautotrophic conditions in a medium which consists essentially of water and minerals.
                    Light and carbon dioxide serve as the only energy and carbon sources.

In-Vitro Translation System 

            JENA:  Cell-free protein expression kit based on the eukaryotic protozoan host Leishmania tarentolae. For target mRNA generation the
                        heterologous T7 RNA polymerase was added to the extracts. To ensure a low background the translation of endogenous host mRNAs
                        is efficiently blocked by an antisense oligonucleotide. 
                        PCR based template preparation  (pdf)
                        Template preparation by cloning into pLEXSY_invitro-2 vector  (pdf)

            NEW ENGLAND BIOLAB:   PURExpress™ In Vitro Protein Synthesis Kit. PURExpress is a novel coupled cell-free transcription/translation
                        system reconstituted from purified components necessary for E.coli translation.
 Due to its reconstitution of recombinant components,
                        PURExpress is essentially free of contaminating exonucleases, RNases, and proteases. Template DNA is not exposed to digestion and
                        target proteins are free of post-translational modifications (glycosylation, phosphorylation, and proteolysis).  Protein synthesis is initiated by
                        the addition of template DNA and is largely complete within one hour.   (pdf)

            PROMEGA  TNT® Quick Coupled Transcription/Translation Systems. Technical Manual. A single-tube, coupled transcription/translation
                        reactions for eukaryotic in vitro translation. Combines RNA polymerase, nucleotides, salts and Recombinant RNasin® Ribonuclease
                        Inhibitor with the reticulocyte lysate solution (60- to 90-minute reaction)
 (pdf-I)  (pdf-II)

             PROMEGA
  TNT® Coupled Reticulocyte Lysate Systems. A single-tube, coupled transcription/translation system.  (pdf)

             PROMEGA   TNT® Coupled Wheat Germ Extract Systems . A one-tube, coupled transcription/translation system   (pdf)

             PROMEGA  Canine Pancreatic Microsomal Membranes. To study cotranslational and initial post-translational processing of proteins.
                        Processing events such as signal peptide cleavage, membrane insertion, translocation, and core glycosylation can be examined by
                        the translation of the appropriate mRNA in vitro in the presence of these microsomal membranes.
 (pdf)

             QIAGEN:   EasyXpress™ Insect Cell Protein Synthesis Handbook. EasyXpress Protein Synthesis Insect Kit. EasyXpress pIX4.0 Vector.
                        For in vitro synthesis of eukaryotic proteins (including membrane proteins) with posttranslational modifications using insect cell lysates
 (pdf)


             QIAGEN:   EasyXpress™ Large-Scale Synthesis Handbook - EasyXpress Protein Synthesis Mega Kit - EasyXpress NMR Protein Synthesis
                        Kits - For large-scale production of proteins by in vitro translation for structural analysis (up to 5 mg of recombinant protein) in E. coli lysates.

                        The EasyXpress NMR Protein Synthesis Kit allows incorporation of the stable-isotope–labeled amino acids threonine, arginine, valine, serine,
                        or lysine.
                        Fifteen further EasyXpress NMR Kits are available for incorporation of any of the remaining 15 amino acids with an isotopic label.
                        The EasyXpress Protein Synthesis Mega Kit allows incorporation of the unnatural, cytotoxic amino acid selenomethionine  facilitating phasing
                        of crystal protein structures and atomic structure determination using X-ray crystallography
    (pdf)

                ROCHE  Rapid Translation System RTS100   (pdf)

                ROCHE_Rapid Translation System RTS500  (pdf)

                ROCHE_Rapid Translation System RTS500 Circular  (pdf)

                ROCHE_Rapid Translation System RTS9000  (pdf)


                ROCHE_RTS_Amino Acid Sampler  (pdf)

                ROCHE_Rapid Translation System GroE Supplement  (pdf) 


 

Entries since September 2006  

 


HOME:
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Selected Protocols
EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
OTHERS


Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mariol@cc.huji.ac.il  Tel: 972-2-6586920  


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