Search

Abstract To optimize the expression conditions for human lactoferrin production, we have constructed the transgenic chlorella with human lactoferrin named as GTD8A1-HLF, the original chlorella was separated from Gurbantunggut Desert in Xinjiang China. To further improve the production of human lactoferrin, a sequential methodology was used to optimize human lactoferrin production by GTD8A1-HLF. First, a screening trial using a Plackett-Burman design was done, and variables with statistically significant effects on human lactoferrin bio-production were identified. These were further optimized by central composite design experiments and response surface methodology. Finally, we found that the maximum human lactoferrin production (52.70 mg/L) was achieved under the following optimized conditions: Initial pH 5.0, NaNO 3 concentration of 0.600 mol/L, FeSO 4 concentration of 0.006 mol/L, and a CuSO 4 concentration of 0.002 mol/L, with the other medium components constituting the basal culture medium BBM. The yield of HLF protein under optimized culture conditions was approximately 4-fold higher than that obtained by using the basal culture medium BBM. The findings are significant for the potential industrial use of GTD8A1-HLF. Highlights • We first successfully obtained the best culture conditions of HLF production in Desert Chlorella, its expression level is 52.70 mg/L under the optimized conditions as initial pH 5.0, NaNO 3 0.600 mol/L, FeSO 4 0.006 mol/L, and a CuSO 4 0.002 mol/L, with the other medium components constituting the basal culture medium BBM. • It was used to analysis and identify results of this paper that Plackett-Burman design, central composite design experiments and response surface methodology, and also help our to predict best component of improved BBM. The yield of HLF protein using the optimized culture medium was approximately 4-fold of that obtained when basal culture medium was used. • The research also provides a new, safe and non-toxic way to produce human lactoferrin, and will hopefully become as commercial production. [ABSTRACT FROM AUTHOR]

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. [ABSTRACT FROM AUTHOR]

Abstract: Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins’ application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense. [Copyright &y& Elsevier]

The Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l -lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 μg of biologically active material that can be recovered in minutes with low concentrations of lysine. Because of their short bed length, spin columns also offer unique advantages, as evidenced by the selective recovery of functional Car9-tagged tobacco etch virus (TEV) protease from a fused and auto-cleaved maltose binding protein (MBP) folding partner that nonspecifically binds to silica in the presence of NaCl. These additional purification modalities should increase the versatility and appeal of the Car9 tag for affinity protein purification. • The Car9 affinity tag enables cheap and rapid protein purification on silica. • DNA purification spin columns and 96-well plates support purification of Car9 fusions. • Spin columns improve tagged protein recovery when nonspecific MBP binding occurs. • These purification modalities increase the versatility and appeal of the Car9 tag. [ABSTRACT FROM AUTHOR]