Bacterial Fold-N-Glow™ Split GFP S11 Plasmid

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Bacterial “Fold ‘n’ Glow” Split GFP S11 Plasmid PDF

Application

Bacterial GFP strand 11 plasmid is a component of the Fold-N-Glow™ Protein Solubility Assay and contains a small (16 aa) GFP fragment for the fusion of the peptide or protein of interest via a flexible linker. Preparation of the s11 plasmid for use in the assay involves generating the s11 fusion protein by cloning the appropriate DNA insert into the S11 vector at the N-terminal. If folded properly or not in the aggregated form, the fusion protein is specifically bound by the s1-10 detection complementation fragment, forming a fluoro phore. This allows for the quantitation of the amount of protein that is properly folded in a given sample as the folding reporter gives a signal directly proportional to the amount of correctly folded protein 1. Misfolding or aggregation of the fusion protein renders the GFP tag inaccessible and prevents complementation, thus preventing fluorescence. Accordingly, misfolded or aggregated proteins are not included in the quantification of the protein of interest. Expressed separately, neither the fusion protein of interest nor the GFP detector (S1-10) is fluorescent.

Features and Benefits

Fluorescent protein (GFP, CFP, or YFP) fusions and split protein tags are widely used for the analysis of protein. These large tags can perturb protein solubility, misfold, and alter the processing of the protein. The split fluorescent protein technology used in the "Fold ′n′ Glow" assay overcomes these problems. The protein tag is a genetically encoded, split fluorescent protein technology, engineered with small, soluble, self-associating fragments. Thus, it is a simple split fluorescent protein system that doesn′t change fusion protein solubility, or require chemical ligation, fused interacting partners, co-expression, or co-refolding. Furthermore, while fluorogenic biarsenical FlaSH or ReASH substrates also overcome these limitations, they also require many other conditions not necessarywhen using the split fluorescent protein technology. The fluorescent protein system is a simple and easy tagging and detection system. These kits may be used to quantify the expression level of the tagged protein, to determine the solubility of a protein, or to determine the solubility of a protein′s domain.

 

Plasmid

(S11): Kanamycin resistance (KanR). BamHI and NdeI restriction sites. Supplied as a 50 μL volume at 2ηg/μL. (S11): Kanamycin resistance (KanR). BamHI and NdeI restriction sites. Supplied as a 50 μL volume at 2ηg/μL.


 Bacterial GFP Strand 11 Plasmid

Product Number 21004003

 PRODUCT INSERT

INTENDED USE: FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES

Bacterial GFP strand 11 plasmid is a component of the Fold-N-Glow™ Protein Solubility Assay and contains a small (16 aa)

GFP fragment for the fusion of the peptide or protein of interest via a flexible linker. Preparation of the s11 plasmid for use

in the assay involves generating the s11 fusion protein by cloning the appropriate DNA insert into the S11 vector at the Nterminal. If folded properly or not in the aggregated form, the fusion protein is specifically bound by the s1-10 detection

complementation fragment, forming a fluorophore. This allows for the quantitation of the amount of protein that is properly

1

folded in a given sample as the folding reporter gives a signal directly proportional to the amount of correctly folded protein .

Misfolding or aggregation of the fusion protein renders the GFP tag inaccessible and prevents complementation, thus

preventing fluorescence. Accordingly, misfolded or aggregated proteins are not included in the quantification of the protein

of interest. Expressed separately, neither the fusion protein of interest nor the GFP detector (S1-10) is fluorescent.

 

REAGENT 

Plasmid (S11) : Kanamycin resistance (Kan ). BamHI and NdeI restriction sites. Supplied as a 50 µL volume at 2 ηg/µL.

Note: The stability of this plasmid is approximately 6 months when stored at -20C. When stored properly, this product is

stable until the date indicated either on the box or each component. Depending on the particular usage requirements, it

may be practical to re-aliquot reagents to smaller working volumes to avoid repeated freeze-thawing or repeated pipetting

from the same vial.

1 NcoI site CCATGG has initiator methionine

2 6HIS tag CATCATCATCATCATCAC amino acids HHHHHH

3 Trombin site amino acids SGLVPPRGS

4 Cloning site NdeI CATATG

5 Frame shift stuffer TAATTAATTAATT

Note that this is a +1 frame shift to make sure S11 doesn’t express unless in-frame construct between NdeI/SpeI or NdeI/BamHI

Typically user can digest NdeI + SpeI and clone that way, or NdeI/BamHI and clone that way. See our Nature methods papers for guidelines on

primer design Cabantous S, Waldo GS (2006) In vivo and in vitro protein solubility assays using split GFP. Nature Methods 3: 845-854.

6 SpeI ACTAGT (expressed as amino acids TS)

7 BamHI GGATCC (expressed as amino acids GS)

8 Linker amino acids DGGSGGGSTS

9 GFP S11 tag

10 stop codon             


 

s-11 Plasmid Gene Map
Materials required, but not supplied:
  • Competent expression cells (BL2/DE3)
  • Kanamycin
  • IPTG (Isopropyl β-D-1-thiogalactopyranoside)
  • LB growth media and plates
  • NdeI And BamHI restriction enzymes
  • Ligation materials
  • Metal affinity column
  • TNG Buffer (50mM Tris pH 7.4, 0.1M NaCl, 10% glycerol)
  • Bovine serum albumin
  • Plasmid Isolation Reagents
  • 96 well plate
  • 4 C refrigerator
  • -20 C freezer
  • Incubator
  • Centrifuge(s) and appropriate size tubes
  • Sonicator
  • Microplate fluorescence reader
  • Vortex mixer
  • Water bath
  • Graduated cylinders and assorted beakers
  • Pipettes and tips
  • Disposable gloves

 


A. Preparation of insert DNA

Prior to performing the assay, carefully read all instructions.

  1. Perform plasmid prep and/or PCR. Use standard materials/protocol not included.
  2. Use NdeI (5’) and BamHI (3’) restriction sites to perform a restriction digest that generates overhangs on the DNA insert. Optional: Purify digest fragment from agarose gel.

B. Preparation of S11 Vector

To ensure that you have a renewable source of plasmid DNA, transform the plasmid vector in an E.coli host
strain.

  1. It is recommended that bacterial frozen stocks be prepared of all transformed plasmids using standard molecular biology techniques.
  2. Purify plasmid DNA for cloning using Plasmid Preparation kits or other techniques (not included).
  3. Perform restriction enzyme digest of the S11 vector using Nde1 and BamH1to prepare the plasmid for the insert DNA. Follow the manufacturer’s instructions for use of the enzymes. Leave sticky ends for over hangs in preparation for ligation.
  4. Optional: Dephosphorylate the digest to decrease non-recombinant background. Use molecular grade calf intestinal or shrimp alkaline phosphatase according to the manufacturer’s directions.
  5. Perform ligation reaction according to manufacturers’ instructions.
  6. Store vector at –20 C until used.

 

REFERENCES

1. Waldo et al. "Rapid protein-folding assay using green fluorescent protein," Nature Biotechnology 17, 691 - 695, July
1999.

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