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Protein Expression and Purification Core FacilityCloning

New SUMO3 fusion vector

Introduction

We have now available a modified expression vector for SUMO3 fusion proteins in pETM11 (with an optimized synthetic sumo3 gene) with appropiate restriction sites at the 3'-end of the gene for sumo3 gene for insertion of any gene of interest.

Human SUMO3 protein can be used as a N-terminal fusion partner (~100 aa, 11.5kDa) enabling high expression yields and good solubility of the target proteins. The fusion partner can be cleaved off by a robust and very specific protease (His6-tagged SenP2 in pETM11 with an optimized synthetic senP2 gene) without any additional amino acids left on your target protein.

The unique feature of this protease is the recognition of the 3-dimensional structure of SUMO3 unlike other proteases that recognize specific amino acid sequences. Thus, unspecific proteolysis of your protein by SenP2 does not occur.

Map and details of the SUMO3 fusion vector

Click to enlarge the map

How to clone you gene into the SUMO vectors

Your target gene can be inserted via the Bam HI-site and one of the restriction sites downstream of it.
In this case, you have to add a N-terminal Ser due to the TCC-codon in the Bam HI-site (if you use the Bam HI-compatible Bgl II-site AGATCT in your insert, it's again a Ser coded by TCT). This Ser will be left in your protein after cleavage with SenP2.

If you don't want any additional amino acids at the N-terminus, you have to use the Age I-site or the compatible sites for Ava I or Xma I (CCCGGG), Bsp EI (TCCGGA) or Ngo MIV (GCCGGC).

In any case, you have to make sure that you have the terminal two Glycine codons of SUMO3 in-frame with your target gene.
Note: The first amino acid after the Glycines must not be Proline! The protease cannot cleave the protein in that case!

Purification of the fusion protein is achieved via the N-terminal His6-tag of SUMO3.
You can also obtain your target protein with a C-terminal His6-tag if you don't add a stop codon to your gene. In this case, please make sure that the second His6-tag is in-frame with your gene. Depending on the 3'-restriction site, you may have to add one additional base.

In the new vector, we have included the gene for EGFP downstream of SUMO3, which results in a vector with following advantages:

  • easy confirmation of double digest of the vector due to release of GFP fragment which is replaced subsequently by your target gene
  • optional N-terminal tagging of your target protein with EGFP by using Sac I and one of the 3'-restriction sites downstream of the EGFP gene (optional with or without C-terminal His6-tag)
  • optional C-terminal tagging of your target protein with EGFP by using Age I and Bam HI

 

Primer design

PCR primers for amplification of your target gene should be designed as follows:
(Note: 6 random additional bases are added to make sure that all restriction enzymes are active, some show poor or no activity without additional basepairs)

Forward primer:

  • Using Bam HI at 5'-end

Alternatively you can replace the Bam HI-site GGATCC with AGATCT (Bgl II)

  • Using Age I at 5'-end

Alternatively you can replace the Age I-site ACCGGT with

CCCGGG (Ava I or Xma I)
TCCGGA (Bsp EI)
or GCCGGC (Ngo MIV)

Reverse primer:

  • with Stop codon

  • without Stop codon (-> C-term His6-tag)

We are happy to assist you with the primer design, if you're not familiar with it.
Please contact: pepcore@embl.de

Example: SUMO3-GFP digested with SenP2

Even with a ratio of 1:1500 (w/w) the SenP2 protease is rapidly digesting the fusion protein at 25°C. At 4°C, a ratio of 1:600 to 1:1500 is sufficient for a 1 h digest, thus requiring only slightly more protease. (For comparison, TEV protease is used at 1:100 or 1:50 ratios for several hours. In the literature, a more than 20-fold higher catalytic activity was confirmed for SenP2 protease compared to TEV protease.*)

 

* Marblestone et al., Protein Science (2006), 15:182-189