Hello colleagues!
I have been to trying to express a human gene but after encountering many challenges, i took a pause to revaluate my strategies. For mammalian genes particularly those that code for enzymes, i know their resulting transcribed mRNA come with a bunch of introns. Do i need to clone cds instead of cloning an entire gene into the plasmid?
yes you must use the coding sequences as human introns will not be processed properly in e.coli if you still have trouble after doing so you might need to codon optimize.
...sure, you should clone the cds instead of the complete gene if you want to express a human gene with intron-exon structure in bacteria, because they do not enable the splice process. Moreover, your plasmid should support prokaryotic gene expression for the inserted sequence. For a high protein expression it may be necessary to optimize the codon structure of the human sequence.
Good luck
To express your protein in bacterial system you have to clone only the coding sequence by making cDNA from respective RNA. If you do not have the RNA then try to amplify the coding sequence by using PCR and run fusion PCR to get your coding sequence for expression and purification.
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