Ca^(2+)-亚氨二醋酸金属离子亲和色谱法吸附内毒素(英文)

Endotoxins(also known as lipopolysaccharides(LPS)) are undesirable by-products of recombinant proteins,purified from Escherichia coli.LPS can be considered stable under a wide range of temperature and pH,making their removal one of the most difficult tasks in downstream processes during protein purification.The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration.Immobilized metal affinity chromatography(IMAC) enables the affinity interactions between the metal ions(immobilized on the support through the chelating compound) and the target molecules,thus enabling high-efficiency separation of the target molecules from other components present in a mixture.Affinity chromatography is applied with Ca2+-iminodiacetic acid(IDA) to remove most of the LPS contaminants from the end product(more than90%).In this study,the adsorption of LPS on an IDA-Ca2+ was investigated.The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal.It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads.The factors such as pH(4.0 or 5.5) and ionic strength(1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than100 EU/mL and 100 000 EU/mL.This new protocol represents a substantial advantage in time,effort,and production costs.

Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body. After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units /mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.

Probing region-resolved heterogeneity of phosphoproteome in human lens by hybrid metal organic frameworks

Phosphorylation plays crucial parts in lenticular biological function.Getting knowledge of region-resolved phosphoproteome contributes to better comprehending the pathogenesis.Here,we prepared the hybrid metal organic frameworks(HMOFs)for probing the region-resolved heterogeneity of phosphoproteome in human lens.1334 phosphosites corresponding to 564 phosphoproteins,1160 phosphosites corresponding to 316 phosphoproteins and 517 phosphosites corresponding to 205 phosphoproteins were identified in capsule,cortex and nucleus,respectively,providing the relatively extensive distribution mapping of phosphorylation in human lens for the first time.The label-free quantification experiments and principal component analysis presented differential expression of phopshoproteins in three subregions.For instance,α-crystallin,β-crystallin and fibrillin-1 closely associated with cataract and Marfan syndrome showed disparate spatial distribution.The preferential phosphoproteins in capsule,cortex and nucleus were involved in cytoskeleton organization,metabolic process and lens development in camera-type eye,respectively.This work first provided a general overview of region-resolved phosphoproteome of human lens.

Identification of zinc-binding peptides in ADAM17-inhibiting whey protein hydrolysates using IMAC-Zn^(2+) coupled with shotgun peptidomics

Food components possessing zinc ligands can be used to inhibit zinc-dependent enzymes.In this study,zinc-binding peptides were derived from whey protein hydrolysates,and their ultrafiltration(>1 and<1 kDa)fractions,produced with Esperase(WPH-Esp),Everlase and Savinase.Immobilized metal affinity chromatography(IMAC-Zn^(2+))increased the zinc-binding capacity of the peptide fraction(83%)when compared to WPH-Esp(23%)and its<1 kDa fraction(40%).The increased zinc-binding capacity of the sample increased the inhibitory activity against the zinc-dependent“a disintegrin and metalloproteinase 17”.LC-MS/MS analysis using a shotgun peptidomics approach resulted in the identification of 24 peptides originating from bovineβ-lactoglobulin,α-lactalbumin,serum albumin,β-casein,κ-casein,osteopontin-k,and folate receptor-αin the fraction.The identified peptides contained different combinations of the strong zinc-binding group of residues,His+Cys,Asp+Glu and Phe+Tyr,although Cys residues were absent in the sequences.In silico predictions showed that the IMAC-Zn^(2+)peptides were non-toxins.However,the peptides possessed poor drug-like and pharmacokinetic properties;this was possibly due to their long chain lengths(5–19 residues).Taken together,this work provided an array of food peptide-based zinc ligands for further investigation of structure-function relationships and development of nutraceuticals against inflammatory and other zinc-related diseases.