Hiroyuki Watabe

Construction of a fusion protein between protein A and green fluorescent protein and its application to Western blotting

Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA‐GFP) migrated at 47 kDa in SDS‐PAGE. However, the non‐fluorescent denatured PA‐GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non‐fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA‐GFP, was used in Western and dot blots. The sensitivity and specificity of the PA‐GFP detection were sufficient for rapid and easy screening.

Purification of recombinant human pepsinogens and their application as immunoassay standards

Human pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable. When these were fused to maltose‐binding protein (MBP), the fusion proteins (MBP‐PGA and MBP‐PGC) were expressed as the major products. Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. MBP‐PGA and the PGA segment obtained by factor Xa digestion (designated as r‐PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t‐PGA). For PGCs (MBP‐PGC, r‐PGC and t‐PGC) also, the specific activities were almost the same. However, the activities of PGCs were about 3‐ to 4‐hold higher than those of PGAs. In PGA and PGC immunoassay systems, r‐PGs (r‐PGA and r‐PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation. From these results, r‐PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems.

A Simple and Rapid Immunoassay System Using Green Fluorescent Protein Tag

A fusion protein between green fluorescent protein (GFP) and neuron-specific enolase (NSE) was expressed in Escherichia coli. The GFP-NSE fusion protein migrated at 62 kDa in SDS-PAGE and retained the fluorescence under non-heating conditions. However, heat-denatured GFP-NSE was non-fluorescent and migrated at 74 kDa corresponding to the theoretical value. This suggests that the special structure of GFP, which is not denatured by SDS, influences its mobility in SDS-PAGE under non-heating conditions. The fluorescence intensity of GFP-NSE was measurable over a wide range by spectrophotometry or densitometry. The competitive immunoassay for NSE was performed using GFP-NSE as labeled antigen. Under our assay conditions, the working range of this system was about 2 -60 ng. This simple and rapid fluorescence immunoassay (FIA) using GFP-tagged antigen may be applicable to many protein markers.

Fluorescence Resonance Energy Transfer-Based Assay for DNA-Binding Protein Tagged by Green Fluorescent Protein

Specific interaction between green fluorescent protein (GFP)-tagged human α- or γ-enolase97-242 (α or γENO97-242) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a “real-time FRET assay.” The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO97-242 and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (Ras) of ENO97-242 mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.

A Highly Sensitive Assay for Proteases Using Staphylococcal Protein A Fused with Enhanced Green Fluorescent Protein

Enhanced green fluorescent protein (EGFP) was fused with staphylococcal protein A (SpA) and used as a substrate for proteases. An SpA-EGFP assay was done in three steps: (i) digestion of SpA-EGFP by proteases, (ii) addition of rabbit IgG immobilized on Sepharose beads, and (iii) measurement of the fluorescence intensity of supernatant. The assay was sensitive enough to measure picogram levels of trypsin and chymotrypsin, and may be applicable to various other proteases as one of the most sensitive methods.

Application of green fluorescent protein-protein A fusion protein to western blotting.

Publisher Summary Green fluorescent protein (GFP) that is isolated from the jellyfish Aequorea victoria noncatalytically produces an intense and stable greenish fluorescence. Aequorea GFP maximally absorbs blue light at 395 nm and emits green light with a peak at 509 nm. GFP is a protein of 238 amino acids with a molecular mass of 27-30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The hexapeptide segment, beginning at residue 64, functions as a fluorescent chromophore formed on cyclization of the residues Ser· dehydro-Tyr-Gly within the hexapeptide, by posttranslational modification. GFP has a unique structure and interesting physical properties—for example, high stability to denaturing reagents or proteases. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence in nonnative organisms in which GFP is expressed. Although GFP is relatively large to serve as a fusion tag, GFP-tagged proteins retain their original functions in many cases. Therefore, GFP has been used as a reporter for gene expression, as a tracer of cell lineage, and as a fusion tag to investigate protein localization and secretion systems in vivo. The reports of GFP fusions to protein A and to streptavidin, and of a simple immunoassay system using a GFP tag also indicate a wide range of in vitro applications. This chapter discusses the construction of a protein A-GFP fusion (PA-GFP) and its use as a labeled antibody-specific ligand in immunoblotting. Immunoblotting requires a labeled antibody or antibody-specific ligand (such as protein A) and a system specifically for detection of the label. Labeling reagents frequently used have been enzymes, such as peroxidase, alkaline phosphatase, and β-galactosidase; gold particles, radioisotopes, and fluorochromes have also been used.

Characterization of recombinant human neuron-specific enolase and its application to enzyme immunoassay.

Human gamma-enolase cDNA prepared by reverse transcriptase-polymerase chain reaction was cloned into the Escherichia coli expression vector pKK223-3. The resulting plasmid, pHTK503, expressed human gamma-enolase as a 46-kDa protein in SDS-PAGE, and in the cells as the active gamma gamma form (designated as recombinant human NSE; R-NSE). R-NSE was purified from E. coli by several chromatographic elutions. Finally, 6.0 mg of R-NSE from 8.1 g cells was purified with a specific activity of 86 units/mg protein. The structural properties of R-NSE were compared with the NSE purified from human brain tissue (B-NSE). The biochemical and enzymatic characteristics were essentially the same, except for the isoelectric point (4.5 for B-NSE and 4.7 for R-NSE). In an NSE immunoassay system, R-NSE and standard NSE were almost equal in reactivity to the anti-NSE antibody. These results indicate that R-NSE can be used as standard assay material.

Modification of human neuron‐specific enolase for application to radioimmunoassay

A recombinant human neuron‐specific enolase (R‐NSE), isolated from Escherichia coli, could not be used in an RIA system because of instability upon labeling. To apply R‐NSE to RIA and to simplify the purification procedure, the N‐ and C‐terminals of R‐NSE were modified by tyrosine‐ and histidine‐tagging, respectively. SY‐NSE, containing one additional tyrosine residue, was obtained from both soluble and insoluble fractions. More derivatives tagged by two or four tyrosine residues were expressed, but only in the insoluble fraction. SY‐NSE and SY‐NSE.H6 (containing six histidine residues at C‐terminal of SY‐NSE) purified from the soluble fraction were applicable to the RIA system, indicating that the addition of a tyrosine residue at the terminal is effective if the antigen is unstable during labeling.

Purification of pepsinogens from human urine and electrophoretic analysis by caseogram print

Pepsinogen (PG) A and C were purified from human urine, and analyzed by a highly sensitive detection method, “caseogram print”. Purification was achieved by a series of conventional chromatographies and FPLC. A relatively large amount (13.2 mg) of PGA was purified from about 20 liters of urine. Purified PGA was separated by a Mono‐Q column into each of its isozymogens. The elution order (PGA‐5, 4+3, 2) corresponded to the order of electrophoretic migration. Although the concentration of urinary PGC was very low, a trace amount was purified and visualized by electrophoresis. The urinary and mucosal PGCs migrated at the same position, and urinary PGC was detected as two isozymogens similarly to mucosal PGC, suggesting that urinary and mucosal PGCs may be essentially identical.