ABSTRACT
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.
Subject(s)
Bacteriophages/genetics , Cell Surface Display Techniques , DNA Restriction Enzymes/metabolism , Nucleic Acid Amplification Techniques/methods , Single-Chain Antibodies/metabolism , Animals , Cloning, Molecular , DNA Restriction Enzymes/genetics , Genetic Vectors/genetics , Mice , Mice, Inbred Strains , Single-Chain Antibodies/geneticsABSTRACT
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.
