Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library.

Efficient refolding of a recombinant abzyme : structural and catalytic characterizations.

Catalytic antibodies are currently being investigated in order to understand their role under physio-pathological situations. To this end, the knowledge of structure-function relationships is of great interest. Recombinant scFv fragments are smaller and easier to genetically manipulate than whole antibodies, making them well suited for this kind of study. Nevertheless they are often described as proteins being laborious to produce. This paper describes a highly efficient method to produce large quantities of refolded soluble catalytic scFv. For the first time, the functionality of a refolded catalytic scFv displaying a ß-lactamase activity has been validated by three approaches: (1) use of circular dichroism to ensure that the refolded had secondary structure consistent with a native scFv fold, (2) development of enzyme-linked immunosorbant assay and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (3) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate.

A promising broad spectrum antimicrobial red fluorescent protein present in silkworm excreta.

The purified silkworm excretory red fluorescent protein (SE-RFP) has exhibited a potent broad spectrum antimicrobial activity. The anti-microbial assays of purified SE-RFP against several pathogenic bacterial (both Gram positive and Gram negative) and fungal strains were performed by agar cup plate method. The minimum inhibitory concentration (MIC) of SE-RFP against pathogenic bacteria and fungi was evaluated by agar dilution technique. The SE-RFP has exhibited highest activity (lowest minimum inhibitory concentration and largest zone of inhibition) against Staphylococcus aureus and Candida albicans among the tested bacteria and fungi, respectively. For the first time, we are reporting here the bioactivity of a red fluorescent protein purified from the silkworm excreta against clinically important bacteria and fungi. The bioactive SE-RFP has two absorption peaks at 280 and 603 nm and, it has exhibited fluorescence emission peaks at 334 and 619 nm upon exciting at 280 and 603 nm, respectively. The SE-RFP being an aqua-soluble, economically feasible and eco-friendly protein, it can therefore be used for the practical applications as an effective antimicrobial agent.

A unique red fluorescent protein of silkworm bearing two photochromic moieties.

A silkworm excretory red fluorescent protein (SE-RFP) having light-dependent activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified. Light was observed to be essential also for the SE-RFP synthesis as it was produced only when silkworms were reared in light. SE-RFP has exhibited a high fluorescence quantum yield of 0.86. The apparent mass of native SE-RFP was about 1100 kDa as analysed by gel filtration chromatography. Two photochromic moieties associated with the SE-RFP, namely tetrapyrrole-I (TP-I) and tetrapyrrole-II (TP-II), were isolated by employing TLC and HPTLC techniques. The purified tetrapyrroles were characterized by UV-absorption, fluorescence, atomic absorption and FT-IR spectral analyses. The molecular masses of TP-I and TP-II were 535 and 870 Da, respectively, as determined by ESI-MS and MALDI-TOF-MS. The molar ratio of TP-I to TP-II was 1.14 : 1.00, and a total of 7.251 micromol tetrapyrroles (TP-I + TP-II) were found to be present per mg of SE-RFP. TP-I and TP-II were identified as chlorophyll derivatives, namely, pyropheophorbide a and pheophytin a, respectively. Hence, the SE-RFP was concluded to be a unique insect red fluorescent protein having two photochromic moieties and potent photobiological activity.