Lorsch Laboratory Methods in Enzymology: Protein Part B

1. Auflage 2014
ISBN: 978-0-12-420179-8
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 539, 240 Seiten

Reihe: Methods in Enzymology

Laboratory Methods in Enzymology: Protein Part B brings together a number of core protocols concentrating on protein, carefully written and edited by experts. - Indispensable tool for the researcher - Carefully written and edited by experts to contain step-by-step protocols - In this volume we have brought together a number of core protocols concentrating on protein

Autoren/Hrsg.

Lorsch, Jon

Leseproben

Chapter One

In Vitro Synthesis of Proteins in Bacterial Extracts

Hani S. Zaher; Rachel Green1    Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA
1 Corresponding author: email address: [e-mail]

Abstract

This protocol describes the methods used to generate protein in a cell-free system derived from E. coli. The in vitro synthesis of protein has been used in studying many ribosome-based gene regulation steps (Gong and Yanofsky, 2001). Such techniques have also been used to study protein–protein, protein–DNA, and protein–RNA interactions, and to produce radiolabeled protein species. More recently, such approaches have been utilized to produce large quantities of toxic proteins for proteomic and structural studies (Yokoyama et al., 2000).

Keywords

Cell-free protein synthesis

E. coli

Centrifugation

French press

Growth and harvest E. coli

In vitro protein synthesis

Optimization of coupled transcription and translation reaction

SDS-PAGE gel

Solutions and buffers

S30 preparation

TCA precipitation

1 Theory

Cell-free protein synthesis systems have been heavily utilized when its in vivo counterparts may fail or prove to be difficult to use. The advantages of in vitro protein synthesis are threefold: the environment of protein production can be readily manipulated by the researchers for optimal conditions, cell viability is not a prerequisite so toxic proteins can be studied, and lastly, large amounts of protein can be produced. Nonetheless, because this is an in vitro system (and may represent an artificial condition), it should be used only if carrying out the experiments in living cells is not feasible. Moreover, because of the complexity of the system, it should be a last resort for protein overexpression.

2 Equipment

French press

Shaking incubator

UV/vis spectrophotometer

High capacity centrifuge

Refrigerated high speed centrifuge

Microcentrifuge

Scintillation counter

Water bath

Polyacrylamide gel electrophoresis equipment

Film developer or Phosphorimager

Side-arm filter flask

Stainless steel filter holder

Erlenmeyer flasks, 250 ml and 4 l

Micropipettors

Micropipettor tips

Pipette aid

Polycarbonate centrifuge tubes, 50 ml

50-ml polypropylene tubes

1.5-ml microcentrifuge tubes

Whatman GF/C glass fiber filters

Autoradiography film or phosphorimager cassette

3 Materials

Acetone

Potassium hydroxide (KOH)

Sodium hydroxide (NaOH)

Bacto tryptone

Bacto yeast extract

Potassium acetate (KOAc)

Potassium glutamate

Ammonium acetate (NH4OAc)

Magnesium acetate (MgOAc)

[35 S]-Methionine (or other radiolabeled amino acid)

Tris base

Glacial acetic acid

HEPES

Sodium dodecyl sulfate (SDS)

Trichloroacetic acid (TCA)

Sodium chloride (NaCl)

EDTA

Phenylmethylsulfonyl fluoride (PMSF)

T7 RNA polymerase

Creatine kinase (CK)

Creatine phosphate (CP)

Pyruvate kinase (PK)

Phosphoenol pyruvate (PEP)

E. coli total tRNA mix

Bovine serum albumin

ATP

GTP

CTP

UTP

Amino acids

L(-)-5-Formyl-5,6,7,8-tetrahydrofolic acid (folinic acid)

cAMP

Dithiothreitol (DTT)

ß-Mercaptoethanol

Polyethylene glycol 8000 (PEG 8000)

40% acrylamide/bisacrylamide (19:1)

3.1 Solutions & buffers

Step 1 2× YT media

Component

Amount

Bacto Tryptone

16 g

Bacto Yeast Extract

10 g

NaCl

5 g

Add water to 1 l. Adjust pH to 7.0 with 5 N NaOH, and autoclave

Buffer A

Add water to 1 l

Step 2

Activation buffer

Add water to 20 ml

Step 3

S30 cocktail

Add water to 1 ml

Amino acids mixture

Make a 10 mM stock of the 19 amino acids (minus the labeled amino acid). Adjust the pH of the mixture to 7.5 using 1 M KOH

4 Protocol

4.1 Duration

Preparation

About 1 day

Protocol

About 2 days

4.2 Preparation

Inoculate a single colony of E. coli into 50 ml of 2× YT medium and grow at 37 °C overnight with shaking (250 rpm). We typically use the BL21 CP strain.

4.3 Caution

Consult your institute Radiation Safety Officer for proper ordering, handling, and disposal of radioactive materials.

See Fig. 1.1 for the flowchart of the complete protocol.

Figure 1.1 Flowchart of complete protocol, including preparation.

5 Step 1 Grow and harvest E. coli for the S30 extract

5.1 Overview

Grow E. coli until the culture reaches mid-logarithmic phase and harvest the cells for the preparation of the extract.

5.2 Duration

3–4 h

1.1 Start three cultures of 1 l each in 4-l Erlenmeyer flasks by inoculating 10 ml of the overnight culture into each flask.

1.2 Grow the culture at 37 °C until they reach mid-log phase (OD600 = 0.9–1.2).

1.3 Quickly chill the cells by immersing the flasks in an ice-water bath.

1.4 Centrifuge the cells, preferably using high capacity rotors (ones that can hold 0.5–1 l of liquid), at 6000 × g, 4 °C for 15 min.

1.5 Decant the spent media and, using a Pipet-aid, carefully resuspend each cell pellet originating from 1 l of culture in 20 ml of ice-cold Buffer A supplemented with 6 mM ß-mercaptoethanol.

1.6 Transfer the resuspended cells into prechilled 50-ml centrifuge tubes before centrifuging at 6000 × g, 4 °C for 15 min.

1.7 Wash the cell pellet one more time with ice-cold Buffer A and centrifuge as before.

1.8 At this stage, the cell pellet can be quickly frozen by immersing it in liquid nitrogen and stored at - 80 °C.

5.3 Tip

The freezing step can affect the overall activity of the extract and, as a result, it is preferable to carry on the next step immediately. Furthermore, the cells should never be frozen for more than 3 days.

See Fig. 1.2 for the flowchart of Step 1.

Figure 1.2 Flowchart of Step 1.

6 Step 2 Preparation of the S30 extract

6.1 Overview

Crack the cells before removing cell debris by centrifugation. Activate the resulting extract to remove endogenous mRNA, and lastly, dialyze against a fresh buffer.

6.2 Duration

16 h

2.1 Carefully resuspend all of the cell pellets in 20 ml of ice cold Buffer A supplemented with 0.5 mM PMSF (add fresh), making sure to avoid frothing. Pass the cells through a syringe to ensure uniformity and to get rid of cell clumps that may clog the French press.

2.2 Crack the cells with a clean, prechilled French press using the manufacturer's instructions.

2.3 Centrifuge the lysate immediately at 30 000 × g, 4 °C for 30 min.

2.4 Decant the supernatant into a fresh centrifuge tube and repeat the centrifugation step. Discard the pellet.

2.5 Decant the clarified supernatant (amber in color) into a graduated cylinder to measure the final volume of the extract.

2.6 Add 0.3 times the volume of Activation Buffer. Transfer into a 50-ml polypropylene tube, cap, and incubate the extract...