Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle.

BACKGROUND: Several fusion tags for separation handle have been developed, but the fused tag for simply and cheaply separating the target protein is still lacking. RESULTS: Separation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli were optimized via precipitation with calcium chloride (CaCl2) and resolubilization with ethylenediamine tetraacetic acid disodium salt (EDTA-Na2). The HanA1-EmGFP absorbing with other three affinity matrix was detected, only it was strongly bound to heparin Sepharose. The separation efficiency of the HanA1-EmGFP was comparable with purification efficiency of the His6-tagged HanA1-EmGFP via metal ion affinity chromatography. Three fluorescent proteins for the EmGFP, mCherry red fluorescent protein and flavin-binding cyan-green fluorescent protein LOV from Chlamydomonas reinhardtii were used for naked-eye detection of the separation and purification processes, and two colored proteins including a red protein for a Vitreoscilla hemoglobin (Vhb), and a brown protein for maize sirohydrochlorin ferrochelatase (mSF) were used for visualizing the separation process. The added EDTA-Na2 disrupted the Fe-S cluster in the mSF, but it showed little impact on heme in Vhb. CONCLUSIONS: The selected five colored proteins were efficient for detecting the applicability of the highly selective hanA1 for fusion separation and purification handle. The fused hanA1 tag will be potentially used for simple and cheap affinity separation of the target proteins in industry and diagnosis.

Controlling expression and inhibiting function of the toxin reporter for simple detection of the promoters' activities in Escherichia coli.

The naturally occurring and mutated promoters inserted into expression plasmids or Escherichia coli chromosome are essential for recombinant protein production and metabolic engineering. Analyzing their activities and screening the promoter libraries require the simple and easy-to-use reporter. Here, we developed a novel and efficient approach to detect the promoter activity, based on E. coli cell growth inhibited by overexpression of bacteriophage ΦX174 gene E product (LyE), but recovered by pre-overexpression of Bacillus subtilis MraY (BsMraY). Under the conditional LyE construct expression in the absence or the presence of the BsMraY, activities of promoters including the reported PT7/lac, Ptac, PBAD, Prha, PhucR, PprpB, Pcum, the wild type and engineered Ptet for leaky and induced expression, the PthrC for auto-induction, and the Pms for constitutive expression were assayed. In one-plasmid coexpression system, the PBAD promoter activity detected using the reporter gene was related to the insertion site. The constructed LyE toxic effects were correlated with toxin expression levels, as determined by the split green fluorescent protein reconstitution. Microscopic analysis showed that cells lysis occurred by the LyE induced with arabinose. Taken together, the toxin reporter construct is a convenient and cost-effective tool to examine the promoter activity in E. coli.

Bacterial production of maize and human serine racemases as partially active inclusion bodies for d-serine synthesis.

Recombinant protein overexpressed in Escherichia coli is often less folded, leading to form the insoluble aggregates also called as inclusion bodies (IBs). IBs are classified as active and inactive ones, and enzyme produced as active IBs is the novel carrier-free immobilized material. In this study, we determined that His6-tagged serine racemase (SR) from maize or human produced as partially active IBs maintained the activities for reversible racemization of l-serine to d-serine but lost the activities for irreversible ß-elimination of both enantiomers, in contrast to the soluble one displaying all activities. Fourier transform infrared spectroscopy analysis showed structural changes between the soluble and insoluble SR. Compared with the soluble SR with attachment of the N-terminal cellulose-binding module via the oriented immobilization of the regenerated amorphous cellulose, the insoluble SR with the fusion of the N-terminal aggregation-inducible tag GFIL8 displayed higher production and usage efficiency, lower leaky capacity, more stability at 4 °C storage with the prolonged time, less sensitivity to the limited proteolysis mediated by trypsin, and higher yield of the synthesized d-serine. These advantages allow the SRs as partially active IBs to synthesize d-serine for medical and agricultural applications.