In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by sitedirected mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding.

The movcmcnt protein of ;rlfal~I mosaic virus wus cxprcsscd in IzrlrrrirlrL ruli tend purified by cation cxchangc chromatography. The purifisd proicin bound singlestranded RPJA caopcntivtly in u biphusic manner. At protein sa~untion. RNAlprotcin cornplcxe (dnigttrtcd 'primnry complexa') wcrc dctatcd by a Ilitroccllulor-ntcntion assay within I min of mixing. bath at 4 and 2X. In contrast, rn incubation of 30 min UI 22.C was nacssary to obtain clectrophorcticolly retarded complcxcs 0ubilizcd camplcxn'), containing a large number of protein moloculcs bound stably to wcli molcculc of RNA. Stabilization did not take plvcc ut 4.C. The nlc of formelien of the primary complclra was rrronSly dcpcn&nt on protein conerntntion. nnd thus appeared limited by u bimolecular inlcnclian. fn contmsl. the rate of stabilization was indcpcndcnt of protein canccntrntion. ruacstiny thttt this process rcmrirtcd oTa rcnrranpmcnt of the primary complcxcs without binding of udditionttl protein molccula, In uBrccmcnt with this rul(g#tion. tht ilmount ofcomplcxcd RNA at equilibrium wus the UI~P when rssnycd by nitroccllulorr: rctcntion nnd by clcctrophorctie rctardrtion. The pasribility thpl these pxuliar kinetics could bccauscd by the prcrensc of Twccn 20 in the incubation mcdiu is discussed. BLII(DE3) cells carrying the rccambinunt pET3d vectors wcrc grown overnight at 37'C in 20 ml LB medium contflining I50 p@ml ampicillin. The culture was diluted twofold with LB ntcdium and another I SO,u#ml of ampicillin WBS uddcd to m&c up for rhc loss due IO the ~.laclumasc rclcxcd during overnight growlh, IPTG (0.4 mM) was added to induce expression of thcchimcric gcnf end the cells were grown for ar,othcr 3 h, The bactcriu wcrc harvcstcd by ccntrifuption and resuspendal in S ml bulTcr A (50 mM Tris+lCl, pH 7.550 mM NKI. 5% glycerol. IO mM EDTA. IO mM 2~mcrraprocthanol) containing 0,4 mylmi lynozymc nnd I mM phcnylmcthylrulfonyi iiucr+!! IPMSF), After 20 min of incr:hation at 37*C. they wcrc rubmittcd to 3 or 4 frccz-th;lw cycles. The inclusion bodies and ccl1 dcbrir wcrc pcllclcd by ccntrifugution at 5.0OO x x for 10 min and rctxtractcd with I.5 ml bulTcr A. then with I,5 ml buifcr B (SO mM Trir-HCI. pH 7.5. IO mM EDTA. 10 mM 20mercaprocthrtnol) containing 1% Nonidct P40 and I mM PMSF, then wtlohcd twice with I.5 ml bulTcr C (SO mM sodium acckttc, pH 5.5, IO mM 2-mcrcaptocthanol). The recombinant protein was solub&cd with I.5 ml bulTcr C contuining 6 M urcil. purified by chromatoyrnphy on cnrboxymcthyl Trisncryl nnd rcnaturcd us dca ssrilxd [II, 11 was atorcdat -80°C in 50%glyccrol, Theconcentration