Part:BBa_K3781006

Strep-tag II + HRV 3C Motif, MocloMania B3

This part codes for a Strep-tag II that is fused to a downstream HRV 3C protease recognition motif.
The Strep-tag II is part of a protein purification system that in its principle it relies on the high affinity binding of the homo-tetrameric protein streptavidin, first isolated from the bacterium Streptomyces avidinii, to the vitamin biotin. The binding of these two biomolecules is one of the strongest non-covalent interactions observed in nature and inspired the creation of many derivatives with even further enhanced binding capabilities. German biotech company IBA Life Sciences developed a synthetic eight amino acid peptide called Strep-tag II which binds to the specifically engineered streptavidin derivative Strep-Tactin with high specificity. Thus, fusing a gene of interest to a Strep-tag II allows for efficient purification of the target protein by affinity chromatography over immobilized Strep-Tactin. The bound target protein can later be eluted with desthiobiotin.

The Strep-tag II encoded in this part is fused to a recognition site for the human rhinovirus 3C protease, a cysteine protease that usually mediates the disruption of host cell transcription during viral cell infection. It binds and cleaves its recognition site with high specifity and, when its motif is fused with a tag, can be used for recovery of tag-free target protein after purification.

size 2.3 kDa

function affinity purification tag

amino acid sequence WSHPQFEK RGSG LEVLFQ/GP

cloning position B3

plasmid backbone pICH41258

Data

We were able to successfully clone this basic part into its respective L0 plasmid backbone and to confirm the integrity of the L0 construct via restriction digest and gel electrophoresis, see Figure 1. Furthermore, we were able to include it into a L1 construct, proving its correct adaptation towards MoClo assembly, see Figure 2.

• 

  Figure 1 | Test digest of L0 B3 constructs
  1 | pICH41258 | BsaI | 2247 + 598 bp
  2 | L0_RBD_B3 | BsaI | 2247 + 675 bp
  3 | L0_3xFLAG_B3 | BstEII + SapI | 1565 + 581 + 212 bp
  4 | L0_Myc_B3 | BstEII + SapI | 1565 + 581 + 167 bp
  5 | L0_GST_B3 | BsaI | 2247 + 684 bp
  6 | L0_Strep_B3 | BstEII + SapI | 1565 + 581 + 167 bp
  7 | L0_8His_B3 | BstEII + SapI | 1565 + 581 + 167 bp
  L | Thermofischer GeneRuler Plus Ladder [bp]
• 

  Figure 2 | Test digest of L1 constructs using HindIII
  1 | L1_sAP_Strep_RBD_Strep8His | 3792 + 3198 + 1005 bp
  2 | L1_sAP_GST_RBD_Strep8His | 3792 + 3198 + 1623 bp
  L | Thermofischer GeneRuler Plus Ladder [bp]

Several L1 constructs have been assembled with this B3 part and successfully transfected into Leishmania, but so far recombinant protein expression could only be detected for L1_sAP_Strep_mCerulean_GST via immunostaining on western blot, see Figure 3.

• 

  Figure 3 | Immunoblot of L1 transfected Leishmania cell cultures | stained against GST
  1 | L1_sAP_RBD_GST | 51.9 kDa
  2 | L1_sAP_Strep_mCerulean_GST | 55.9 kDa
  n.c. | negative control | Leishmania culture transfected with empty L1 expression vector
  L | Thermofischer PageRuler Protein Ladder [kDa]
  1. AB | gt anti-GST | 1:10,000
  2. AB | rb anti-gt HRP | 1:2,000
  

So far, no L1 construct assembled with L0_Strep_B3 has been successfully purified. Furthermore, to this day, detection against the Strep-tag II using anti-Strep antibodies has remained unsuccessful. Unlike most other antibodies, the Strep-antibodies available in our working group were coupled to alkaline phosphatase, requiring a different detection mechanism than horseraddish peroxidase-mediated chemiluminescence. We suppose that expression signals were too weak for visual alkaline phosphatase detection using BCIP and NBT. In the future, acquisition of HRP-tagged anti-Strep-tag II antibodies might facilitate Strep-based detection. We are working hard on realizing this in order to qualitatively validate the functionality of our basic part.

The MocloMania collection

This basic part is part of the MocloMania collection, the very first collection of genetic parts specifically designed and optimized for Modular Cloning assembly and recombinant protein expression in the protozoan parasite Leishmania tarentolae.

Are you trying to express complexly glycosylated proteins? Large antibody side chains? Human proteins that require accurate post-translational modification? Then Leishmania might be just the right organism for you! Leishmania tarentolae’s glycosylation patterns resemble those of human cells more closely than any other microbial expression host, while still delivering all the benefits of microbial production systems like easy transfection and cultivation. So instead of relying on mammalian cell lines, try considering Leishmania as your new expression host of choice!

Our MocloMania collection will allow you to easily modify your protein of choice and make it suitable for downstream detection and purification procedures - all thanks to the help of Modular Cloning. This cloning system was first established by Weber et al. in 2011 and relies on the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequence, hereby generating four nucleotide overhangs. Every basic part in our collection is equipped with a specified set of overhangs that assign it to its designated position within the reading frame. These so-called cloning positions are labelled B2-B5 from upstream to downstream. By filling all positions with the basic parts of your choice, you can easily generate variable genetic constructs that code for the fusion protein of your desire.

We furthermore provide a specifically domesticated Leishmania expression vector, named weird_plex, which will package your fusion construct into a functional transcriptional unit that is optimized for high expression in Leishmania.

The best part? Because of the type IIS restriction properties and the specifity of the generated overhangs, restriction and ligation of your construct can all happen simultaneously in a simple one-step, one-pot reaction. This will safe you a lot of time and frustration in your cloning endeavours!

Do we have your attention? In the table below you can find some basic information on how our cloning system, along with most other MoClo systems, is set up. Please feel free to check out our wiki to find more information on Leishmania and Modular Cloning as well as to understand how this basic part integrates into our part collection. See you there!

MOCLO | Important nomenclature and parameters

Level	What does this level contain?	antibiotic resistance	Enzyme used for ligation
L0	The foundation to every MoClo construct which are basic genetic units, such as coding sequences, promoters, terminators	spectinomycin	BbsI
L1	Several L0 parts assembled into a functional transcriptional unit, e.g. consisting of promoter, coding region and terminator	ampicillin	BsaI
L2	Multiple transcriptional units added into one multi-gene construct, e.g. a protein of interest fused to a selection marker	kanamycin	BbsI

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]