Yield was 30 mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both Tozasertib supplier with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wildtype apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies. (C) 2011 Elsevier Inc. All rights reserved.”
“N-terminal fusion tags

that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site

as the overlap region allows the fusion of different 17 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator find more (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C(3)G(3) repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter

constructs are then used as templates see more for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolyl cis-trans isomerise B (PpiB) fusion tag which produces 1 mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the “”stress-responsive proteins”". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches. (C) 2011 Elsevier Inc. All rights reserved.”
“In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin alpha’ subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed alpha’E.