title: Approximated maximum adsorption of His-tagged enzyme/mutants on Ni2+-NTA for comparison of specific activities.
abstract: By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yuanli
authors.initials: Y
authors.lastname: Li
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Gaobo
authors.initials: G
authors.lastname: Long
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Hu
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yiran
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Deng
authors.initials: D
authors.lastname: Tan
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.affiliation: Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Medical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China. Electronic address: liaofeish@yeah.net.
authors.forename: Fei
authors.initials: F
authors.lastname: Liao
mesh_terms: D000327:Adsorption; D004789:Enzyme Activation; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry*; D050505:Mutant Proteins; D009532:Nickel / Q000737:chemistry*; D011993:Recombinant Fusion Proteins
chemical_list: D004798:Enzymes; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D009532:Nickel; D008239:Lysine
keywords: 6His-tagged alkaline phosphatase/mutants; Approximation; Cell lysates; Ni(2+)-NTA-magnetic-submicron-particles; Saturation adsorption; Specific activities