A histidine-rich fusion tag enables real-time monitoring of recombinant protein expression by Pauly reaction-based colorimetric assay.

Commercially available recombinant expression systems always use fusion tags to facilitate target protein purification and SDS-PAGE analysis followed by Coomassie Brilliant Blue (CBB) staining is the classical method to validate the expression level of target protein, which is time-consuming, although not very laborious. Previously, we found that a histidine-rich elastin-like polypeptide (HRELP) tag could make its fusion proteins being quickly and specifically stained with Pauly's reagent. In this study, we designed a Pauly reaction-based colorimetric assay to real-time monitoring of the expression level of recombinant protein tagged HRELP and found that the absorption value of post-induction E. coli cells stained with Pauly's reagent correlated well with both the band intensity of the target protein from Pauly's reagent-stained and CBB-stained gels. Moreover, we found the colorimetric assay could also be helpful to roughly estimate the expression efficiency by using a poly-histidine-tagged protein, which has only 1.17% histidine residue. In our opinion, Pauly reaction-based colorimetric assay could significantly shorten the time to validate the over-expression of recombinant protein tagged with either HRELP or poly-histidine. And HRELP seemed to be an ideal fusion tag for it can not only facilitate protein purification but also simplify protein detection.

Soluble expression of mature Rhizopus chinensis lipase in Escherichia coli and enhancement of its ester synthesis activity.

The production of membrane-associated lipase from Rhizopus chinensis (RCL), which has a high ester synthesis activity and important potential applications, is difficult in heterologous expression system such as Escherichia coli and often leads to the formation of inclusion bodies. Here, we describe the soluble expression of mature RCL (mRCL) using maltose-binding protein (MBP) as a solubility-enhancing tag in the E. coli system. Although the MBP-mRCL fusion protein was soluble, mRCL was insoluble after removal of the MBP tag in E. coli BL21 (DE3). Using E. coli BL21 trxB (DE3) as an expression host, soluble mRCL was obtained and expression conditions were optimized. Furthermore, the ester synthesis activity of soluble mRCL was increased by detergent treatment and was found to be 3.5 and 1.5 times higher than those of the untreated enzyme and naturally occurring enzyme, respectively. Overall, this study provides a potential approach for producing active and soluble forms of eukaryotic lipases in a heterologous E. coli expression system.

A histidine-rich elastin-like polypeptide functions as a quickly detectable and easily purifiable protein fusion tag.

Although many protein fusion tags have been developed for recombinant protein production to improve protein yields or facilitate purification, determining the expression and purification of the fusion protein still remain to be a time-consuming and laborious procedure. In this work, we designed a histidine-rich elastin-like polypeptide (HRELP) fusion tag and found that it could be efficiently expressed in E. coli cells and specifically stained with Pauly's reagent in a couple of minutes post SDS-PAGE analysis. Moreover, in Pauly's reagent-stained polyacrylamide gels, only the bands of HRELP fusion proteins were yellow and could be clearly visualized with little background. Furthermore, both HRELPs and HRELP20-BMP2 fusion protein could be purified by a method of pH shift-mediated inverse transition cycling (ITC). In our opinion, the HRELP established in this study may be considered as a multifunctional protein tag which could make its fusion proteins being quickly detected by Pauly staining and simply purified by pH-triggered ITC in addition to having the potential to sustained release its fusion proteins.

Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression.

Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein's pI is suggested.

Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor.

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence reported to date. In the present work, we developed NanoLuc as a novel quantitative protein fusion tag for efficient overexpression in Escherichia coli and ultrasensitive bioluminescent assays using human leukemia inhibitory factor (LIF) as a model protein. LIF is an interleukin 6 family cytokine that elicits pleiotropic effects on a diverse range of cells by activating a heterodimeric LIFR/gp130 receptor. Recombinant preparation of the biologically active LIF protein is quite difficult due to its hydrophobic nature and three disulfide bonds. Using the novel NanoLuc-fusion approach, soluble 6×His-NanoLuc-LIF fusion protein was efficiently overexpressed in E. coli and enzymatically converted to monomeric mature LIF. Both the mature LIF and the NanoLuc-fused LIF had high biological activities in a leukemia M1 cell proliferation inhibition assay and in a STAT3 signaling activation assay. The NanoLuc-fused LIF retained high binding affinities with the overexpressed LIFR (Kd = 1.4 ± 0.4 nM, n = 3), the overexpressed LIFR/gp130 (Kd = 115 ± 8 pM, n = 3), and the endogenously expressed LIFR/gp130 (Kd = 33.1 ± 3.2 pM, n = 3), with a detection limit of less than 10 receptors per cell. Thus, the novel NanoLuc-fusion strategy not only provided an efficient approach for preparation of recombinant LIF protein but also provided a novel ultrasensitive bioluminescent tracer for ligand-receptor interaction studies. The novel NanoLuc-fusion approach could be extended to other proteins for both efficient sample preparation and various bioluminescent quantitative assays in future studies.