Editing MBP fusion protein expression in CAG629 and CAG597

== NEB's E.coli strain CAG629 and CAG597 for pT7A3-MBP or pT7A30-MBP fusion protein expression<br>  ==

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E.coli strain CAG597 has a temperature dependent suppression of an amber codon within the gene for RpoH (sigma factor). The result is that growth at 37C or higher does not result in the transcriptional activation of either sigma factor or the dozens of protease/chaperones upregulated by sigma. This in theory can increase the solubility of recombinant proteins expressed in this cell line. 

One drawback is that the strain is harder to transform than many others and requires methylated plasmid DNA. NEB’s literature says one way to methylate the plasmid is to first transform your plasmid into e.coli TB-1, grow overnight, then do a plasmid prep. I found that the widely used DH5a worked just fine also. 

If unsure you can check for methylation by incubating the plasmid with a restriction enzyme like Mbo1 which is very methylation sensitive. Your original unmethylated plasmid probably has 30+ Mbo1 sites and should look like a DNA ladder after digestion versus little or no digestion of your methylated plasmid. 

For purists a triple selection plate with tet, strep and your plasmid’s selectable marker should be used. Otherwise keep the triple selection on until after transformation has been accomplished, then just use your plasmid selection (ampicillin in my case) while expressing your recombinant protein at 37C for about 3 hours. Grow the cells to an approximate OD600 of 0.7 before inducing with IPTG, 1mM.<br> 

C41(DE3) for toxic protein<br> 

Since the strain C41(DE3) was derived from BL21(DE3) it should allready be deficient for lon and ompT proteases. If including protease inhibitors in your lysis buffer does not alleviate your problem, you can try E.coli host strains that lack other proteases besides lon and ompT. KS100 for example lacks a periplasmic protease (Tsp/prc). <br> 

=== Strain CAG629 is best for expressing unstable proteins in cytoplasm. <br>  ===

A suspension of E. coli strain K12 CAG629 which has been grown in LB medium and brought to 50% glycerol. E. coli K12 CAG629 has the rpoHam allele (sometimes referred to as htpRam) which encodes an amber mutant form of the sigma factor for the heat shock response. Grow by streaking out on an LB plate and incubating at 30°C.<br>Genotype: F- lacZ(am) pho(am) lon supC(ts) trp(am) rpsL rpoH(am)165 zhg::Tn10 mal(am)<br>Properties and Usage Back to Top<br>Storage Temperature: -20°C<br>Antibiotic Resistance: tet, str 

Growth Media: LB<br>Growth Temperature: 30 °C<br><br> 

The strain has a temperature-sensitive amber supressor tyrT(supCts), so upon incubation at 37°C or 42°C the strain does not suppress the heat shock sigma factor RpoH. Thus it becomes deficient in expression of the heat shock proteins. Since a number of heat shock proteins affect proteolysis, this can increase the stability of foreign proteins expressed in E. coli. CAG629 is EcoK r+m+, so plasmids have to be modified (i.e., come from an m+ strain such as TB1, JM83, JM107, etc.) in order to get transformants; the transformation frequency is 10-100X down from other common strains used for recombinant DNA work.

== <br>E. coli K12 CAG597 for periplasmic expression<br> ==

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A suspension of E. coli strain K12 CAG597 which has been grown in rich LB medium and brought to 50% glycerol. E. coli K12 CAG597 has rpoHam allele (sometimes referred to as htpRam) which encodes an amber mutant form of the sigma factor for the heat shock response. Grow by streaking out on an LB plate and incubating at 30°C.<br>Genotype F- lacZ(am) pho(am) tyrT[supC(ts)] trp(am) rpsL(StrR) rpoH(am)165 zhg::Tn10 mal(am)<br><br>Storage Temperature: -20°C<br>Antibiotic Resistance: str, tet

Growth Media: LB<br>Growth Temperature: 30 °C <br>

The strain has a temperature-sensitive amber supressor TyrT [supC(ts)] , so upon incubation at 37°C or 42°C the strain does not suppress the heat shock sigma factor RpoH. Thus it becomes deficient in expression of the heat shock proteins. Since a number of heat shock proteins affect proteolysis, this can increase the stability of foreign proteins expressed in E. coli. CAG597 is EcoK r+m+, so plasmids have to be modified (i.e., come from an m+ strain such as TB1, JM83, JM107, etc.) in order to get transformants; the transformation frequency is 10-100X down from other common strains used for recombinant DNA work<br>

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== Degradation of MBP-fusion<br> ==

It is likely that the fusion protein is degraded, leaving a stable MBP-sized breakdown product. In this case, try using a protease deficient host. NEB Express is Lon- and OmpT-.&nbsp; For cytoplasmic expression, the most protease deficient strain is CAG629 (NEB #E4125S) – it is also, however, the most difficult to work with. CAG597 (NEB #E4123S) is another good alternative. For '''periplasmic''' expression, the most protease deficient strain is '''CAG597''' (NEB #E4123S); KS1000 (NEB #E4128S) and UT5600 (NEB #E4129S) might be worth trying as well. The CAG strains are difficult to transform, and often require electroporation to introduce the fusion plasmid. <br>

[[Category:Protocols]]