EVALUATION OF CELL FREE EXPRESSION OF PROTEINS FOR USE AS INTERNAL STANDARD REFERENCE MATERIALS

Faith A. Hays, Johanna Camara, David Bunk

The development of clinical reference measurement procedures and materials at NIST has predominantly been for electrolytes and small organic hormones and metabolites.  A more recent focus on protein reference materials and measurements at NIST has been necessary to ensure traceable, high-quality measurements for the multitude of critically important clinical protein biomarkers.  The route to standardized clinical protein measurements is, for reasons of molecular size, molecular complexity, and analyte concentration, often more challenging than for electrolytes and small organic molecules and NIST’s efforts in this area are still very immature.  However, the need for standardized clinical laboratory measurements of proteins is likely to expand substantially from worldwide research efforts in biomarker discovery. 

The use of stable isotope labeled peptides and mass spectrometry for quantification of digested protein biomarkers has been widely reported in the literature.  The main limitation of this approach is that the stable isotopically labeled peptides are introduced into the sample after purification and digestion of the protein biomarker.  Therefore, the isotopically labeled peptides cannot account for losses of protein during purification and variability of the digestion process.  A more ideal internal standard would be a stable isotope labeled protein added to the matrix and co-purified and digested with the protein of interest. 

In this approach, in vitro cell free expression of the protein of interest is applied using stable isotope-labeled amino acids to ensure a mass shift of the peptides when analyzed by mass spectrometry.  A commercially available cell free expression kit from Roche was evaluated and it was determined using fluorescence that the kit produced 4-9µg per 50µl reaction of the standard green fluorescent protein (GFP).  A histidine tag was inserted into the DNA construct to aid in purification of the proteins.  The His-tag based purification of the GFP was shown to effectively purify the protein using one-dimensional SDS-PAGE.    

 

Author: Faith Hays

Mentor: David Bunk

CSTL, Analytical Chemistry Division 

Building 227, Mail Stop 8392, Room B146

P: (301) 975-3689 

F: (301)977-0685  

faith.hays@nist.gov

Not a Sigma XI member