Production and Purification of the Native Saccharomyces cerevisiae Hsp12 in Escherichia coli

Hsp12 is a small heat shock protein produced in many organisms, including the yeast Saccharomyces cerevisiae. It has been described as an indicator of yeast stress rate and has also been linked to the sweetness sensation of wine. To obtain a sufficient amount of protein, we produced and purified Hsp12 without tag in Escherichia coli. A simple fast two-step process was developed using a microplate approach and a design of experiments. A capture step on an anion-exchange salt-tolerant resin was followed by size exclusion chromatography for polishing, leading to a purity of 97%. Thereafter, specific anti-Hsp12 antibodies were obtained by rabbit immunization. An ELISA was developed to quantify Hsp12 in various strains of Saccharomyces cerevisiae. The antibodies showed high specificity and allowed the quantitation of Hsp12 in the yeast. The quantities of Hsp12 measured in the strains differed in direct proportion to the level of expression found in previous studies

Purification process of recombinant monoclonal antibodies with mixed mode chromatography

An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs. (C) 2015 Elsevier B.V. All rights reserved

Selective Tuning of Elastin-like Polypeptide Properties via Methionine Oxidation.

We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)(n) with variable lengths (n = 20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127 mg/L culture. After digestion of the fusion proteins by enterokinase, the ELF moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4 degrees C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELF of low molecular mas

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)(n) with variable lengths (n = 20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127 mg/L culture. After digestion of the fusion proteins by enterokinase, the ELF moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4 degrees C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELF of low molecular mass. (C) 2015 Elsevier Inc. All rights reserved

Selective Tuning of Elastin-like Polypeptide Properties via Methionine Oxidation

We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media

Use of the human hepcidin gene to build a positive-selection vector for periplasmic expression in Escherichia coli

Recombinant proteins are often produced in the periplasm of Escherichia coli because this facilitates the purification process. The oxidizing environment favors the formation of disulfide bridges. We showed that the periplasmic expression of the human hormone hepcidin 25 (Hep25) fused to the maltose binding protein (MBP) resulted in cell death. This toxicity was not observed when MBP-Hep25 accumulated in the bacterial cytoplasm, or when Hep25 was addressed to the periplasm without the MBP tag. We then modified the periplasmic expression vector pMALp2E to create pMALp2EH, a positive-selection vector with Hep25 as counterselection gene. (C) 2016 Elsevier Inc. All rights reserved

Production and purification of recombinant human hepcidin-25 with authentic N and C-termini

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long-sought hormone that regulates iron homeostasis in mammals. Recombinant human Hepcidin-25 (Hepc25) was expressed in Pichia pastoris using a modified version of the pPICZ alpha A vector. Hepc25 was then purified by a simple two-step chromatographic process to obtain 1.9 mg of soluble recombinant human Hepc25 per liter of culture at 96% purity. The sequence of Hepc25 and the presence of four disulfide bridges were confirmed by mass spectrometry analyses, and the recombinant Hepc25 exhibited antibacterial activity. This protocol of production and purification is the first step toward the production of human Hepc25 at a greater scale. (C) 2015 Elsevier B.V. All rights reserved

Development of anti-chloro 192 tyrosine HDL apoA-I antibodies for the immunodiagnosis of cardiovascular diseases

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