pET-28a-Cramoll

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	rCramoll was obtained by heterologous expression using the expression vector pET-28a-Cramoll and the strain E. coli Rosetta (DE3), followed by purification using Sephadex G-75 column as described by Varejão et al. (2010).

Protocol tips
rCramoll was obtained by heterologous expression using the expression vector pET-28a-Cramoll and the strain E. coli Rosetta (DE3), followed by purification using Sephadex G-75 column as described by Varejão et al. (2010).

Publication protocol

rCramoll was obtained by heterologous expression using the expression vector pET-28a-Cramoll and the strain E. coli Rosetta (DE3), followed by purification using Sephadex G-75 column as described by Varejão et al. (2010). A 24 full factorial design was used to study the effects of some factors on the expression level of soluble rCramoll: Cell density at induction measured as optical density at 600 nm (OD600; 0.5 or 4.0), isopropyl-β-D-thiogalactoside (IPTG) concentration (0.05 or 1 mM), time of induction (2 or 22 h), and temperature during the induction (37 or 15°C). The combinations resulted in eight different expression experiments, which were performed in triplicate. Three repetitions at the center point level (OD600: 2.25; 0.55 mM of IPTG, 26°C and 11.5 h) were also performed to determine if there is a non-linear relationship between the variables and the responses. After the expression experiments, the E. coli cells were harvested by centrifugation and disrupted by sonication on ice (Varejão et al., 2010). Soluble and insoluble fractions were separated by centrifugation and the presence of recombinant Cramoll was analyzed by denaturing SDS-PAGE electrophoresis. The protein concentration was quantified on scanned gels using the Quantity One software (Bio-Rad). Purified rCramoll was applied on gels at 5 μM as a standard for the quantification.

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