Dear all, I am sorry to bother you with two off-topic questions.
Question one: I have solved a pig mitochondrial protein crystal structure whose molecular weight is about 95kd. However, I do not have the sequence information of this protein. It is weird that I could not blast its sequence in pig genome. Because pig sequences of known proteins are very similar to their human homologous, I used this protein's human homologous sequence to fit into electron density. Currently, I am planning to sequence the full length sequence of the protein. However, I was not able to successfully extract mRNA from pig liver using Qiagen RNeasy kit (I only tried once). Does any body have ideas about how to extract mRNA from pig live (or animal tissue in general)? Are there any other database I can search for pig protein sequence information? Question two: At the same time, I was trying to express the human homologous of the protein in E.Coli so that I would be able to do some functional studies. I tried pET21b and pET28a vectors. Protein was not expressed when pET28a construct was used but expressed in insoluble form with pET21b vector construct. I tried different strains, lower IPTG concentrations (down to 0.1 mM), lower temperatures (down to 15 degree), adding cofactor, detergent extraction and so on. I even truncated the first 17 residues. I was not able to obtain soluble human protein at all. Does any body have experiences working with human mitochondrial proteins? There is another question which might be very naive. I saw people publishes important rat protein crystal structures although sequences of their human homologous are known. Does it mean that rat proteins are more stable than human ones? Any suggestions would be greatly appreciated. Thank you for your suggestions in advance! Best wishes Wei Yong
