Determination of Protein Synthesis Yield with PURExpress (E3313, E6800, E6840, E6850)

Introduction

Using the equations below, one can calculate the yield of protein synthesized in the reaction. The calculations do not differentiate full-length protein from truncated products and as such, all translation products contribute to the calculation of yield. Prior to using the equations, it is necessary to have determined the number of picomoles of both labeled and unlabeled methionine in the reaction, the number of counts produced by no template (background), target protein (TCA-precipitable) and the total counts in a reaction.

picomoles of Met: 

unlabeled = 0.3 mM in reaction = 300 pmol/μl x 25 μl rxn = 7,500 pmol 

labeled = 2 μl of ³⁵S-Met (15 mCi/ml, 1,000 Ci/mmol) per rxn = 2 μl x 15 mCi/ml = 30 μCi x μmol/1 x 10⁶ μCi = 3 x 10^-5 μmol = 30 pmol 

total = 7,500 pmol unlabeled Met + 30 pmol labeled Met = 7,530 pmol Met 

Total counts = total cpm per 5 μl control x (reaction volume/5) 

Specific Activity = Total counts / pmoles methionine (labeled and unlabeled) 

Met incorporation (pmoles) = [(TCA ppt cpm-background cpm) x total reaction volume/5] / Specific Activity 

pmoles of protein = pmoles of incorporated Met / # of Met residues in target protein 

Yield of protein (μg) = pmoles of protein x MW of protein / 10⁶

Example of Calculation: 

DHFR: 
17,998 Daltons, 5 methionine residues 

CPM's Measured: 
1.12 x 10⁷ total, 5 μl aliquot
1.8 x 10⁴ background
1.95 x 10⁶ TCA ppt 

Total Counts = 1.12 x 10⁷ cpm x 5 = 5.6 x 10⁷ cpm/25 μl rxn 

Specific Activity = 5.6 x 10⁷cpm / 7530 pmol = 7437 cpm/pmol

Methionine Incorporation = (1.95 x 10⁶ cpm – 1.8 x 10⁴ cpm) x 5 / 7437 = 1297 pmol Met 

pmoles DHFR = 1297 pmol Met / 5 Met/DHFR 

Yield (μg) = 259.4 x 17,998 / 10⁶ = 4.67 μg/25 μl rxn x 40/ml = 187 μg/ml