Prbbbb:small scale expression v1

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Overview

This protocol describes how to express small protein amounts from 10 ml culture for expression screening. The constructs are supposed to be under control of the T7 promoter. BL21 strains contain an inducible T7 polymerase.

Culture Growth

day 1

1. prepare LB+Amp (or appropriate antibiotic) Agar plates with Glucose added to 1% final concentration
   • (prepare stock of sterile-filtered 20% Glucose)
2. transform expression construct into BL21DE3 cells using normal protocol
3. plate transformation on LB + 1% Glucose + Antibiotic
   • grow over night @ 37°C but avoid overgrowing clones -- 13 or 14 h are best

day 2

1. inoculate 4 clones from each transformation plate into 3 ml LB + 1% Glucose
2. grow over night @ 37 °C

day 3

1. prepare glycerol stock of each clone -- use 10 % final glycerol concentration
   • backup for later as different clones of one construct can have varying expression strengths
   • glycerol content is a bit lower than normal; decreases risk of plasmid loss
2. inoculate 10 ml 2xTY + 1% Glucose from over night culture at 1:100 dilution
3. incubate shaking @ 37°C until OD600 reaches 0.5 (usually 2-3 h)
4. induce expression: add IPTG to final concentration of 0.1 - 1 mM (0.5mM as a start)
5. let induced cultures grow (A) 3 h @ 37 and (B) over night @ 20°C
6. measure OD600 of 37°C cultures
7. aliquot 10 ml into 5 x 2ml tubes
8. pellet by centrifugation
9. store pellet @ -20°C

day 4

1. measure OD600 of 20°C cultures
2. aliquot 10 ml into 5 x 2ml tubes
3. pellet by centrifugation
4. store pellet @ -20°C

Lysis and Analysis

Cell lysis by sonication

1. resuspend pellet in 500 µl extraction buffer
   • extraction buffer: add 1 "complete mini" Protease inhibitor tablet to 10 ml PBS
2. transfer into 1.5 ml Eppendorf tubes
3. sonicate
   • miniprobe: apply 2 x 20s intermediate strength burst
   • ultrasound bath: 5 min @ intermediate strength
   • check solution and adapt length accordingly

or: Enzymatic cell lysis

1. prepare BugBuster (Novagen) buffer (1ml 10x buffer + 9ml ddH20 + 1 tablet Complete, Mini (protease inhibitor, Roche))
2. resuspend pellet in 300 µl BugBuster buffer
3. shake 20 min @ RT

Gel electrophoresis

1. centrifuge 1 min @ max. r.p.m. in table-top centrifuge to remove intact cells
2. transfer supernatant
3. centrifuge 30 - 40 min @ 20.000 g (@ 4°C, cold room)
   • supernatant = soluble fraction
   • glassy pellet = membrane bound and inclusion bodies
4. soluble fraction:
   • prepare 2 samples (a) and (b) of 20µl each:
   • normalize samples to 20µl of culture with lowest OD after induction (e.g. directly take 2 x 20µl from lowest ID supernatant but take less from the others and fill up to 20 µl)
   • add 5µl 5x sample buffer with SDS
   • denature 5 min @ 95°C
5. insoluble fraction:
   • resuspend in 50µl 5x sample buffer by repeated up/down pipetting
   • denature 5 min @ 95°C
   • prepare 2 samples (a) and (b) of about 5µl each:
   • normalize exact amount according to OD after induction

1. load samples (a) on gel A for Commassi staining
2. load samples (b) on gel B for Western blot