I transformed a strain of lactobacillus using electroporation to introduce a constitutive expression vector (P-erm is the promoter) expressing a recombinant protein. The construct was first introduced into an E. coli strain to make stocks of the plasmids. The western blot results indicated the expression of this protein using both an anti-his antibody and a specific antibody.
The construct was then introduced into lactobacillus and a positive clone was selected using an antibiotic. I confirmed the presence of the construct using primers flanking the inserted genes and also confirmed that it was free of mutations using Sanger sequencing. However, I could not see any bands in WB (using whole cell lysate, soluble fraction isolated after sonication, and inclusion bodies solubilized in 8M urea) but a faint band of the correct size was present in both Coomassie blue staining and Ponceau staining (for the soluble fraction).
What do the results mean? Any suggestion on modulating the culturing condition for better protein expression?
Hello! There could be some issue with the codon usage? Did you check the GCUA, https://gcua.schoedl.de/ The usage frequency of some of the codons in your transcript could be too low. Is this strain of lactobacillus optimized for recombinant protein expression? Are there restriction enzymes that are active in this strain? E. coli for protein expression has been optimized, perhaps there is some necessary modifications that this strain is lacking? Good luck!!
Below is the Ponceau S staining result. There's quite a strong band in lane 1 & 5-7 yet they could not be stained.
I also wonder what is with the strong staining at the bottom of the membrane. Does it mean that my proteins were degraded?
I added 1mM PMSF before sonication in ice. Do i have to use a higher concentration?
Have you tested the cDNA to check for expression from the promoter in your target bacteria species?
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