Protein Engineering Protocols - Mycobacteriology research center

224 Fujita et al.3. Streptavidin and GST genes.4. RTA gene.5. Gene III gene of fd phage derived from pCANTAB 5E (Amersham Biosciences,Piscataway, NJ).6. Oligonucleotide primers and linkers.7. T7 Ampliscribe kit (Epicentre Technologies, Madison, WI).8. Cap-structure analog (New England Biolabs, England).9. DNase I (Epicentre Technologies, Madison, WI).10. RNeasy mini kit (QIAGEN, Hilden, Germany).11. Flexi ® Rabit Reticulocyte Lysate system (Promega, Madison, WI).12. Recombinant RNasin ® Ribonuclease Inhibitor (Promega).13. Binding (or washing) buffer: 140 mM NaCl, 2.7 mM KCl, 10.1 mM Na 2HPO 4,1.8 mM KH 2PO 4, 0.04% Tween-20, 2% Block Ace (Dainippon PharmaceuticalCo.; Japan), and 5 mM MgCl 2, pH 7.3. Stable at –20°C for up to 1 mo.14. Ligand beads for the selection.15. Biotin agarose (Sigma, St. Louis, MO) and glutathione sepharose 4B (AmershamBiosciences).16. Elution buffer: 1 M NaCl and 50 mM EDTA.17. ReverTra Dash ® (Toyobo, Japan).18. KOD Dash ® (Toyobo).3. MethodsThe methods described here outline the construction of the expression plasmidand the cycle of RIDS for the selection of functional protein.3.1. The Construction of Expression Plasmid for RIDSFirst, DNA constructs encoding the T7 promoter, protein library, linker, RTA,and spacer should be prepared to construct RIDS (Fig. 2A), as described inSubheadings 3.1.1. to 3.1.3. Although it is possible, theoretically, to prepare thedouble-stranded DNA sequence using a polymerase chain reaction (PCR) method,we recommend making the DNA construct using plasmids, because it may be difficultto connect various motifs (namely, the T7 promoter, protein library, linker,RTA, and spacer) using PCR without nonspecific amplification. In this section, wedescribe the procedure of plasmid construction for RIDS. DNA manipulationswere performed using standard recombinant DNA methods to construct the plasmid,and are not described here in detail because of space limitations.3.1.1. Construction of the pLRS Expression PlasmidAt first, we prepared the universal plasmid, pLRS, encoding the T7 promoter,linker, RTA, and spacer as the DNA construct for RIDS (Fig. 2A). The plasmidis based on the expression system derived from the pET30a plasmid (Novagen).We can construct the protein library for RIDS easily by insertion of a cDNAlibrary or random library at the XbaI/NheI sites in the pLRS plasmid.