I want to clone have two proteins cloned into pPROexHtb+ (which has one cloning site). I want to express them as a protein complex. My question is would you generally put a stop codon after first gene of interest and start with a start codon for the next gene of interest? Or - would you clone two genes of interest one after the other without any stop/start codon?
You need the stop codon of course, if you don't plan to have only one protein chain containing both proteins (or one being out of frame if you are not careful). You can find some info on di-cistronic expression here: https://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/dicistronic_cloning/index.html
Alternatively you can do the fusion approach of expressing both proteins in the same ORF but a protease site (e.g. TEV) inbetween and ideally the TEV protease as a third protein, if there is a problem with stoichiometry of the partners. With this approach you can make sure that you get a 1:1 ratio.
It would be simpler however to have the genes on two vectors (with different antibiotic resistance) as described here: https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/co-expression/
Note that it is generally recommended to have different orignis of replication for the two vectors in co-expression experiments, but it is not really necessary if you are growing under normal conditions for less than 24h.
You could use a vector from the pEtDuet series, which carry two cloning sites preceded each by a T7 promoter. This would avoid problems of low experssion due to polarity on the downstream gene.
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