Case Studies - Baculovirus Expression
1. Project evaluation and high secretory expression level (6mg/L)
Customer’s initial request was to synthesize the gene in question and to clone it into our BV vector for intracellular expression. The expected yield was 0.1-1 mg of tagged protein with over 70% purity. Based on a thorough sequence analysis, we designed a new strategy, which was approved by the customer, touse the protein's native signal peptide to drive secretion. In the end, we obtained 3 mg of protein with a purity level over 80% from 0.5 L SF9 CM. The protein's identity was further confirmed by MALDI-TOF.
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Fig. SDS-PAGE Analysis of target protein purified by SP column
Lane 1-8: Eluted fractions with 1 M NaCl in 20 mM PB, pH 5.0 |
2. Tag-free protein purification
After protein expression, the conditioned medium was dialyzed against buffer, and loaded onto SP column. The predicted size of the target protein was 78 kDa. The SDS-PAGE results showed that proteins of the same size as the target had been obtained in lanes 12, 13, 14.
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Fig. SDS-PAGE Analysis of target protein purified by SP column
Lane 1: The condition medium
Lane 2-14: Eluted fractions with gradient concentration of NaCl from 0 to 500 mM in 20 mM PB, pH 5.0 |
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