Preparation, Biochemical Analysis, and Structure Determination of Methyllysine Readers

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. Author manuscript; available in PMC: 2018 Oct 16.

Published in final edited form as: Methods Enzymol. 2016 Feb 1;573:345–362. doi: 10.1016/bs.mie.2015.12.005

Troubleshooting

Problem	Potential Cause and Solution
Leaky expression seen in preinduced samples	Switch to a cell line that suppresses background expression such as pLysS or pLysE cells
No expression seen in postinduction sample	The full-length protein is either not expressing or is being degraded in the cell. (1) Rare codons in the gene can lead to severely truncated transcripts. Check for the presence of rare codons and if needed either switch to a compensatory cell line such as BL21-CodonPlus or Rosetta2 or codon optimize the gene. (2) The rate of translation or induction time needs to be altered. Optimize the concentration of IPTG, the induction temperature and length of induction and analyze yield by SDS-PAGE. (3) Expression is low in the chosen media. Switch media.

Problem

Potential Cause and Solution

Leaky expression seen in preinduced samples

Switch to a cell line that suppresses background expression such as pLysS or pLysE cells

No expression seen in postinduction sample

The full-length protein is either not expressing or is being degraded in the cell. (1) Rare codons in the gene can lead to severely truncated transcripts. Check for the presence of rare codons and if needed either switch to a compensatory cell line such as BL21-CodonPlus or Rosetta2 or codon optimize the gene. (2) The rate of translation or induction time needs to be altered. Optimize the concentration of IPTG, the induction temperature and length of induction and analyze yield by SDS-PAGE. (3) Expression is low in the chosen media. Switch media.