Editing S30 E. coli extract (section)

== Protocol 7 ==

=== In Vitro Translation of scFv Antibody Fragments. ===

In vitro translation in an E. coli S-30 system was performed according to Chen and Zubay (16) with several modifications. Translation was carried out for 10 min at 37°C in a 110-μl reaction mixture that contained the following components: 50 mM Tris⋅HOAc at pH 7.5, 30 mM NH4OAc, 12.3 mM Mg(OAc)2, 0.35 mM each amino acid, 2 mM ATP, 0.5 mM GTP, 1 mM cAMP, 0.5 mg/ml E. coli tRNA, 20 μg/ml folinic acid, 100 mM KOAc, 30 mM acetylphosphate (17), 1.5% polyethylene glycol 8000, 33 μg/ml rifampicin, 1 mg/ml vanadyl ribonucleoside complexes (VRC), 23 μl of E. coli MRE600 extract (16), and 90 μg/ml mRNA.

=== Affinity Selection of Ribosome Complexes and Isolation of RNA. ===

The translation was stopped by adding Mg(OAc)2 to a final concentration of 50 mM and cooling on ice (18). The samples were diluted 4-fold with ice-cold washing buffer [50 mM Tris⋅HOAc, pH 7.5/150 mM NaCl/50 mM Mg(OAc)2/0.1% Tween 20] and centrifuged for 5 min at 4°C at 10,000 × g to remove insoluble components. Microtiter plates coated with hemagglutinin-transferrin conjugate were prewashed with ice-cold washing buffer, and the supernatant from the centrifuged translation mixture was applied (200 μl per microtiter well) and the plate was gently shaken for 1 hr in the cold room. After five washes with ice-cold washing buffer, the retained ribosome complexes were dissociated with ice-cold elution buffer (100 μl per well; 50 mM Tris⋅HOAc at pH 7.5/150 mM NaCl/10 mM EDTA/50 μg/ml E. coli tRNA) for 10 min in the cold room, and released mRNA was recovered by precipitation with ethanol or by isolation using the Rneasy kit (Qiagen).

=== Reverse Transcription–PCR and in Vitro Transcription. ===

Reverse transcription was performed using Superscript reverse transcriptase (GIBCO/BRL) according to the supplier’s recommendation. PCR was performed using Taq DNA polymerase (GIBCO/BRL) in the presence of 5% (vol/vol) dimethyl sulfoxide (4 min at 94°C, followed by 3 cycles of 30 sec at 94°C, 30 sec at 37°C, and 2 min at 72°C, followed by 10 similar cycles at 60°C instead of 37°C, 20 similar cycles at 60°C instead of 37°C with elongation at 72°C prolonged by 15 sec per cycle and finished by 10 min at 72°C). PCR products were analyzed by agarose gel electrophoresis and purified from the gel and reamplified, if the amount and quality was not sufficient, or directly used for transcription without additional purification. In vitro transcription was performed as described (19).

=== In Vitro Coupled Transcription–Translation and RIA. ===

After the fifth round of ribosome display, PCR products were cloned into the vector pTFT74 (20). In vitro coupled transcription–translation in the S-30 E. coli system was performed using 50 μg/ml plasmid DNA and conditions similar to those described above with the following modifications. A coupled transcription–translation was carried out for 30 min at 37°C, and the reaction mixture was supplemented with T7 RNA polymerase at 2000 units/ml and 0.5 mM UTP and TTP. The mixture contained [35S]methionine at 50 μCi/ml (1 μCi = 37 kBq) and each amino acid except methionine at 0.35 mM. After translation, the reaction mixture was diluted 4-fold with PBS and bound to immobilized hemagglutinin peptide in a microtiter well. After 60 min of incubation with gentle shaking, microtiter wells were washed five times with PBS containing 0.1% Tween 20, and bound radioactive protein was eluted with 0.1 M triethylamine. Eluted protein was quantified in a scintillation counter.
[[Category:Antibodies]]