Overexpression, purification, and characterization of recombinant T4 gene 32 protein22-301 (g32P-B).

摘要: Gene 32 protein (g32P), the replication accessory from bacteriophage T4, is a zinc metalloprotein which binds with high cooperativity to single-stranded (ss) nucleic acids. The basic N-terminal 21 amino acids (termed "B" domain) required for highly cooperative (omega approximately 500) binding of g32P monomers ss As part our studies systematically evaluate structural features B domain important binding, homogeneous source noncooperatively = 1) and devoid contamination by native needed. Herein, we describe large-scale overexpression purification recombinant lacking tryptic (residues 1-21), designated g32P-B, as well its physiochemical acid properties. G32P-B readily purified soluble fraction Escherichia coli BL21 (DE3) transformed plasmid pT7g32-B.wt contains g32P-B coding sequences under inducible transcriptional control T7 RNA polymerase. Anion exchange, ssDNA-cellulose phenyl-Sepharose chromatographies give rise free contaminating acid. Recombinant has expected primary structure stoichiometric Zn(II). It also globular shown 1H NMR spectroscopy, hydrodynamic measurements, ability selectively remove carboxyl-terminal "A" form trypsin-resistant g32P-(A + B) DNA-binding core fragment. Quantitative experiments poly(dT) (0.05 M NaCl, pH 8.1, 20 degrees C) show that all equilibrium isotherms can be fit omega 1 Kobs 5.2 (+/- 1.6) x 10(5) M-1, moderate electrostatic component energy, delta log Kobs/delta log[NaCl] -3.0 +/- 0.2. Under identical solution conditions, derived expected, but an 80-fold higher apparent affinity, 4.0 2.0) 10(7) detectable enhanced salt sensitivity, -3.9 0.3. concentration raised, relative difference in between gradually reduced such extrapolation log-log plots Na+ standard state gives similar within experimental error. Qualitatively observations are found upon ribohomopolymer, poly(U).(ABSTRACT TRUNCATED AT 400 WORDS)