Home About us Contact
|
Home About us Contact | ||
|
|
||
One-step Purification (one-step + purification)
Selected AbstractsExpression of Secreted His-Tagged S -adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and ImmobilizationBIOTECHNOLOGY PROGRESS, Issue 1 2008Yunxing Luo S -Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S -adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomycescerevisiae was successfully produced at high level (,200 mg/L) by the recombinant methylotrophic yeast Pichiapastoris. The secreted His6 -tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 °C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl -Met and ATP. [source] Enzyme Replacement Therapy for Murine Hypophosphatasia,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008José Luis Millán PhD Abstract Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5,-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6 -dependent seizures. There is no established medical treatment. Materials and Methods: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2,/,), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, ,CT, and histomorphometry. Results:Akp2,/, mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. Conclusions: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2,/, mice. [source] Preparation and a simple one-step purification of [His1 -mono- 125I-Tyr10,Nle27]-hGHRH(1-32)-NH2JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 1 2002Janos Gardi Abstract A one-step purification of [His1 -mono- 125I-Tyr10,Nle27]-hGHRH(1-32)-NH2, prepared using chloramine-T, by HPLC with isocratic elution is described. The labeled GHRH analog was suitable for GHRH receptor binding assays. Copyright © 2002 John Wiley & Sons, Ltd. [source] Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteinsJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2008Dexian Dong Abstract Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins. Copyright © 2008 John Wiley & Sons, Ltd. [source] Design of Affinity Tags for One-Step Protein Purification from Immobilized Zinc ColumnsBIOTECHNOLOGY PROGRESS, Issue 1 2000Richard S. Pasquinelli Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichiacoli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions. [source] | ||||||||||