Invitrogen Bac to Bac Expression System
Invitrogen Bac to Bac Expression System
Invitrogen Bac to Bac Expression System
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Cloning in<strong>to</strong> pFast<strong>Bac</strong> HT A, B, and C<br />
Introduction<br />
The pFast<strong>Bac</strong> HT vec<strong>to</strong>r is supplied with the multiple cloning site in three<br />
reading frames (A, B, and C) <strong>to</strong> facilitate cloning your gene of interest in frame<br />
with the N-terminal 6xHis tag. See the recommendations below and the diagrams<br />
on pages 12-14 <strong>to</strong> help you design a cloning strategy.<br />
Cloning<br />
Considerations<br />
The pFast<strong>Bac</strong> HT vec<strong>to</strong>rs are fusion vec<strong>to</strong>rs. To ensure proper expression of your<br />
recombinant protein, you must:<br />
• Clone your gene in frame with the initiation ATG at base pairs 4050-4052. This<br />
will create a fusion with the N-terminal 6xHis tag and the TEV protease<br />
cleavage site<br />
• Include a s<strong>to</strong>p codon with your insert<br />
The production of recombinant proteins requires that your insert contain a<br />
translation initiation ATG. Generally, transfer vec<strong>to</strong>rs that contain intact<br />
polyhedrin (PH) leader sequences (e.g. pFast<strong>Bac</strong> vec<strong>to</strong>rs) may yield higher levels<br />
of expression than vec<strong>to</strong>rs that contain interrupted leader sequences. Protein<br />
translation can initiate at the mutated ATG (ATT) upstream of the multiple cloning<br />
site; however, initiation from this site is inefficient and generally does not interfere<br />
with expression and detection of recombinant protein.<br />
continued on next page<br />
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