I cloned an insect amylase gene in pICZ alpha-A vector. Confirmed that the gene was in the right reading frame by sequencing. I used the EasyComp kit for preparing the competent cells of X33 and got a few clones which on plate assay showed clearance on starch plates. I expressed one of the clones in BMMY medium and got expression. Expression worked only the first time. After that, all attempts to express the protein using the same clone and same conditions have failed! Can somebody advice vme as to what could have gone wrong!
Thanks in advance.
Problem in expressing a protein in Pichia pastoris
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