Materials and Reagents

1. UV-Star^® PS microplate (96 well) (Greiner Bio-One GmbH, catalog number: 655801 )
   
2. 0.22 µm filter
   
3. Escherichia coli BL21 (DE3)
   
4. Trizma^® base (Sigma-Aldrich, catalog number: T1503 )
   
5. Purified recombinant His-tagged M1Pase
   Note: This protein was produced in Escherichia coli BL21 (DE3) containing the recombinant pFO4_ M1Pase vector, as described by Groisillier et al. (2010). This recombinant protein was purified by affinity chromatography using a HisPrep FF 16/10 column (GE Healthcare) onto an Äkta avant system (GE Healthcare). The complete purification protocol is described in details in Groisillier et al. (2014).
   
6. MgCl₂ (Sigma-Aldrich, catalog number: M8266 )
   
7. MilliQ water
   
8. Malachite green phosphate assay kit (Gentaur, catalog number: POMG-25H )
   Note: Malachite green phosphate assay protocol is available at https://www.bioassaysys.com/Datasheet/POMG.pdf.
   
9. Examples of chemicals to be tested to assess substrate specificity
1. D-mannitol-1-phosphate (Sigma-Aldrich, catalog number: 92416 )
   
2. D-fructose-1-phosphate (Sigma-Aldrich, catalog number: F1127 )
   
3. D-glucose-1-phosphate (Sigma-Aldrich, catalog number: G9380 )
   
4. D-mannose-6-phosphate (Sigma-Aldrich, catalog number: M3655 )
   
5. D-glucose-6-phosphate (Sigma-Aldrich, catalog number: G7879 )
   
6. D-fructose-6-phosphate (Sigma-Aldrich, catalog number: F3627 )
   
10. NaCl (Sigma-Aldrich, catalog number: 71380 )
    
11. 1 M Tris-HCl (pH 7.5) (see Recipes)
    
12. 100 mM MgCl₂ (see Recipes)
    
13. 5 M NaCl (see Recipes)
    

Equipment

1. NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, model: NanoDrop 2000 )
   
2. Safire2 UV spectrophotometer microplate reader (Tecan Trading AG)
   

Software

1. Hyper32 (Informer Technologies, http://hyper32.software.informer.com/)
   
2. Microsoft Excel
   

Procedure