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Cloning Strategies for Expression Vector Preparation

Due to the diverse nature of proteins the requirements for the optimal expression conditions have to be evaluated for each protein individually. The most important features in this process are the expression host (E. coli, mammalia, yeast and insect cells), the promoter and the affinity tags (Strep-tag®, 6xHistidine-tag, GST-tag and FLAG-tag).

With StarGate we developed a cloning system which allows a fast and convenient screening for the optimal expression features. Rapid and standardized subcloning of an arbitrary gene into a wide collection of expression vectors (Acceptor Vectors) is enabled without time consuming planning.

StarGate Cloning

General information on StarGate cloning

StarGate for fast protein expression cloning

StarGate has been developed for the rapid systematic screen of the optimal expression (regarding expression host and/or promoter) and purification (regarding the fusion tag) system for a given gene of interest (GOI).

There are two possible cloning procedures:

Direct Transfer Cloning

The direct transfer cloning procedure is recommended for proteins with known purification conditions. With this strategy the PCR product of a certain GOI can directly be transferred within a one-step-reaction into the appropriate Acceptor Vector.

“Two-step-cloning” via pENTRY-IBA

StarGate cloning via the pENTRY-IBA vector provides a tool for the systematic and rapid screen of the best working expression conditions for a certain GOI, where nothing is known from literature. After initial pENTRY cloning, the GOI in the resulting Donor Vector can be easily transferred by a simple one-tube reaction into a multitude of Acceptor Vectors providing different features.

Acceptor Vectors

IBA Acceptor Vectors provide different genetic surroundings for optimal protein expression and purification. These include different purification tags, promoter for a variety of host cells, signal sequences etc.

Direct Transfer Cloning

Direct Transfer Cloning

The Direct Transfer Cloning procedure is recommended in cases the optimal expression and purification system is already known. In this procedure, a PCR product containing the gene of interest can be directly inserted into the appropriate Acceptor Vector without the need for prior generation of a Donor Vector.

Cloning Procedure

In a first step the PCR fragment is generated using the respective forward (CF) and reverse (CR) primer extending the gene of interest (GOI) with the corresponding integration sites. This PCR product is in the second step integrated into the appropriate Acceptor Vector resulting in the final expression construct, the so called Destination Vector. The formation of the correct Destination Vector is monitored via blue-white screening on LB-Agar plates containing X-Gal. The validated Destination Vector can be directly used for transformation or transfection of the expression host.

Two-step-cloning via pENTRY

“Two-step-cloning” via pENTRY-IBA

The "Two-step cloning" approach via the Donor Vector is recommended in cases where nothing is known about the expression of the respective gene of interest. This procedure allows rapid and highly efficient subcloning of an arbitrary gene into a variety of expression vectors in parallel. In a first step the gene is cloned into the pENTRY vector to generate the Donor Vector. In a second step the gene can be transferred from this Donor Vector into Acceptor Vectors, which provide different genetic surroundings (e.g. tags, promoters and signal sequences). The resulting vector is called Destination Vector and is placed into the respective expression host.

Detailed Cloning Procedure via pENTRY-IBA

Step 1: Entry Cloning for Donor Vector generation

In the first step, the gene of interest (GOI) will be equipped at both termini with combinatorial sites and the LguI recognition sites, which are important for oriented insertion of the PCR fragment into pENTRY-IBA51. This is done by PCR using a proofreading polymerase.
Recombination of the PCR product with the Entry Vector at the combinatorial sites (red and orange) leads to generation of the Donor Vector. This step involves loss of the LguI restriction sites (dark orange with arrowheads), making the recombination reaction unidirectional and thereby highly efficient.

In the resulting Donor Vector the same combinatorial sites are now flanked by the Esp3I recognition sites (light blue), thereby enabling  a highly efficient and specific StarGate® gene transfer process into Acceptor Vectors in a similar manner.

Step 2: Transfer Cloning (Destination Vector Generation)

In the transfer reaction step, the GOI is transferred from the Donor Vector into an Acceptor Vector. The Acceptor Vectors provide the desired genetic surroundings (i.e. affinity tag, promoter (prom), additional signal sequences, etc.). In cases the optimal expression and purification conditions of the GOI are unknown, a variety of different Acceptor Vectors should be chosen.

The transfer from the Donor Vector into the Acceptor Vector is assisted by Esp3I as restriction enzyme and T4 DNA Ligase. In the resulting Destination Vector, the GOI is placed under control of the selected promoter (prom), allowing GOI expression in the selected expression host. In this example, an affinity tag (green) is fused to the C-terminal end of the GOI expression product.

Special features provided by StarGate Cloning

Cloning via Gene Synthesis

Gene synthesis – the easy way to generate your StarGate Donor Vector

In the past decade, prices for gene synthesis services have become more and more favorable. Therefore, gene synthesis, offered by a wide range of scientific service providers, is a fast and easy alternative to create a StarGate Donor Vector containing your GOI (gene of interest). Compared to standard cloning procedures (via PCR, restriction enzyme digestion, purification steps, ligation), gene synthesis is a comfortable way to generate a StarGate Donor Vector, offering the opportunity to optimize the codon usage of your GOI to gain improved protein expression in your particular host.

How to generate a StarGate Donor Vector by using a gene synthesis service:


  1. Enter the sequence of your GOI without start and stop codon
  2. Select specific codon optimization for your final host. Add the Esp3I recognition site and the respective combinatorial sites:
    Start: 5'-AGCGCGTCTCCAATG
    Stop: 3'-GGGAGGAGACGCGCT
  3. Choose a standard cloning vector (offered by the service provider) containing an antibiotic resistance gene for kanamycin
Fusion of two or more proteins into an Acceptor Vector

Generate fusion proteins or construct artificial operons for multiple gene expression in bacterial or mammalian cells

Fuse genes from any Donor Vector in any combination to make single fusion proteins or even to construct artificial operons for multiple gene expression in bacterial or mammalian cells. The StarGate® Fusion Cloning system allows easy and fast fusion of two different genes of interest (GOI-1 and GOI-2) by an intergenic region (IR). Only two sequential StarGate subcloning reactions with dedicated Fusion Vectors are required. The procedure can be repeated to fuse more than two GOI's.

 

In the first reaction, the two GOIs need to be transferred into special fusion vectors by two separate transfer reactions performed in parallel. The GOI that is intended to be positioned upstream in the final construct is cloned into a pNFUSE-IBA derivative, carrying the IR, while the second GOI is placed into pCFUSE-IBA11, which is always the same irrespective of the desired IR. The cloning into the fusion vectors is driven by the Esp3I restriction enzyme.

In the second reaction, GOI1 and GOI2 cloned in the Fusion Vectors are assembled in a directed manner using LguI as restriction enzyme into pENTRY-IBA51 by a second one-tube reaction.

Intergenic regions for tandem gene expression

The StarGate Fusion Cloning system allows easy and fast fusion of two different genes of interest (GOI-1 and GOI-2) by an intergenic region (IR). Such an intergenic region, e.g. codes for an amino acid linker sequence directly connecting two proteins (LINK11/12). Alternatively, it may include a Shine Dalgarno (SD) or an IRES site for the construction of synthetic operons in bacterial or mammalian cells, respectively. This allows independent expression of both proteins, whereas the linker sequence produces a large fusion protein of both GOIs.

StarPrimer D'Signer

Innovative software for StarGate Entry Cloning Primer

The StarPrimer D'Signer 3.0.0.3 software facilitates the design of primers for the initial ENTRY cloning step based on its ease of use.
The software is also very well suited for the design of primers in combination with a site-directed mutation approach. In this case, it is used to design primers for introducing mutations into your gene of interest, which then is transferred into a StarGate pENTRY vector to create a Donor Vector carrying the mutated gene of interest.
This software not only helps with the primer design, it also guides step by step through the whole PCR process.

To use the new StarPrimer D'Signer 3.0.0.3 software please click StarPrimer D'Signer or on the picture (right)
 

Requirements to run StarPrimer D'Signer Software: 

  • Installation of Microsoft Silverlight is necessary to run the StarPrimer D´Signer software.
  • System requirements:
    • For PC (Win 2000 SP4, Win XP SP 2/3, Windows Vista and Windows 7)
    • For Apple Macintosh ( Intel Processor and MAC OS X 10.4.11 )
  • Browser should be current version of Internet Explorer or Firefox.


strep-tag@iba-lifesciences.com

 

PTO Protected Cloning Primers

For StarGate cloning we recommend the use of 3’ phosphorothioate (PTO) protected oligonucleotides which are protected against the exonuclease activity of Proof Reading Polymerases. These Primers can be directly ordered at IBA’s Custom Oligo division, which offers an excellent custom service for synthesis of a wide range of high quality nucleic acid specialties at competitive prices. 

Classic Cloning

pASK-IBA and pEXPR-IBA cloning vectors for expression in E. coli and mammalian cells that provide different protease cleavage sites or chloramphenicol resistance (E. coli).

Cloning system

The polylinkers of the expression vectors carry the restriction sites BsaI (isoschizomer Eco31I) and BsmFI (New England Biolabs, MBI Fermentas) which allow the precise fusion of the structural gene with the vector-encoded functional elements (including Strep-tag®II, 6xHistidine-tag and, depending on the vector, OmpA-signal sequence, start codon, protease cleavage site or stop codon. This is easily achieved by adapting both ends of the coding region of the structural gene via PCR.
To avoid the incorporation of base substitutions, PCR should be performed with a proof-reading DNA polymerase such as Pfu (Stratagene). 3' phosphorothioate-protected primers should be used in order to avoid 3'-5' degradation by the proof-reading activity.