# Determination of Protein Synthesis Yield with PURExpress (E6800)

## 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