S-Tag Rapid Assay Th e Se Ta g rapid ass ay is ba se d on th e recons titution of ribonucl eolytic (RNase S) activity. A sampl e containing the target pro- tein is add ed to a buffer containing purified S-prot ein a nd th e ribonucl ease s ub s tr ate poly(C) . After a bri ef incubation the reac- tion is stopp ed with trichloroacetic acid a nd the resulting precipitate removed by cent:ri- fu ga tion. Activity is measur ed by reading th e ab s orban ce of th e s up e rn ata nt at 280nm , which increases as th e poly(C) is brok en down into acid- s olubl e nucleotides by the enzyme (3 ). In the presence of excess S-prot ein th e level of RNa se S ac tivity is dir ec tly proportion a l to th e amount of recombinant prot ein prese nt in the sample. Ty pi ca l assay pr o fil es ar e s hown in Fi g ur e 1 . A lin ea r sig nal wa s obtained during a 5 minut e incubation at 37°C. In thi s exp erime nt , 2fll of c rud e tran sla tion mix and 2pl of a 11100 dilution of E. coli lysed with 1 % SDS were used as samples. Appropriat e blanks serve as controls for any endogenous RNa se activity, which app ears to be minimal und er the condition s of th e Rapid Assay. By comparing dIe ab sorbance profile of a known s tand a rd ( provid ed in th e kit ), th e mol ar con ce ntration of th e SeTag t arget prot ein can be calc ulat ed. Unlik e amino ac id in corporation a ss ay s ( both radioa c tiv e and non - radio ac tiv e) , SeTag Rapid Assay is independent of pro- tein s iz e, amino acid composition a nd en- dogenous amino acid pool size. It, therefore, re pr esent s a mu ch mor e acc urat e me thod for measuring in vitro tran slation effi cien- ci es . Th e method also is extremely versatile for meas uring prot ein e xpre ssion in cells, since it can be applied to bodl soluble a nd in solubl e prot eins. Up to 10]11 of a 1/100 dilution of 6M ur ea or g uanidine HCI ca n be add ed to the assay with lime effect ( dat a not shown). Multiple sa mples can easily be screened for expr ession levels by preparing crud e e xtra cts of who le ce ll s in 1 % SDS. Since the ass ay wi ll detect as little as 20fmo l tar ge t pr otein in a 5 minut e in c ubation , even po o rl y e xpr esse d protein s ca n be meas ur ed with a high degree of accur ac y. Typi cally, Iml of induced c ultur e provides enough material for over 1000 SeTag Rapid Assays or 100 SeT ag Western Blots. SeTag Western Blot Western blotting of SeT ag prot eins provides a seco nd m ea ns of det ec tion and al lows vis ualization of prot e in int e grity. Th e SeTag Weste rn Blot Kit is ba sed on th e intera c tion be twe en th e SeTag se qu ence ruld S-protein:biotin or enzyme conjugates. Colorimetric substrate s ar e included which allow as l ittl e as 250pg of tar get protein to be visualiz ed using a 30 minute protocol. Figur e 2 shows a time course of induction of a' nd a recombinrult antibod y cloned in pET-30b(+ ). The SeTag western blot shows high specificity for target pro- teins ( and th eir amino-t erminal- containing breakdown products ) with very low back- ground s taining of oth er E. coli prot eins. Th e gels al so contain Novagen 's Pe rfe ct Prot ein ™ markers, a set of 7 proteins \villi pr ec ise mole cular we ight s a t c onv e ni e nt int e rval s ( 15 , 25, 35, 50 , 75 , 100 , and 150kd ). The markers are known amowlt s of defin ed recombinant proteins containing dle SeTag and thu s se rv e as precise int e rnal s tandards for SeTag western blots. S-Tag Affinity Purification Th e high affinity intera c tion be tw ee n S-protein and SeTag also can be applied to purifi ca tion of target prot eins. Th e SeTag Purification Kit contains S-protein immobi- lized on agaro se bead s to ac hi eve rapid , continued on page 6 S-Peptide in Protein Fusion Systems: History and Significance Jin-Soo Kim and Ronald T. Raines - Department of Biochemistry, University of Wisconsin-Madison R ibonuclease A (RNase A) catalyzes the cleavage of RNA. Almost forty years ago, Fred Richards and coworkers discovered that the protease subtilisin prefers to cleave a single peptide bond in native RNase A (1). The product of this cleavage, ribonuclease S (RNase S) , consists of two tightly-associated fragments : S-peptide (residues 1-20) and S-protein (residues 21-124) . Although neither fragment alone has any ribonuclease activity, RNase S has enzymatic activity similar to that of intact RNase A. Richards and Harold Wyckoff determined the structure of crystalline RNase S by x-ray diffraction analysis (2). The S-peptide fragment of RNase A has played an important role in the history of biochemistry. Before molecular biologists were able to use recombinant DNA technology to explore protein structure-function rela- tionships , organic chemists synthesized analogs of S-peptide and studied their complexes wi th S-protein . These studies provided much information on the role of individual residues in RNase S. Most significantly, Chris Anfinsen and coworkers found that only residues 1-15 of S-peptide were necessary to form a fully functional complex with S-protein (3) . This shorter fragment is called "S15" (or '' SeTag™'' by Novagen) and its complex with S-protein is called "RNase S:' The detection, immobilization, and purification of proteins is idiosyn- cratic and can be problematic . Fortunately, these processes can be gener- alized by using recombinant DNA technology to produce fusion proteins in which target proteins are fused to carrier polypeptides (4). The affinity of the carrier for a specific ligand enables the facile detection, immobilization ( continued on page 6) •