Journal of Chemical Technology and Applications

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Affinity Chromatography Journals

We have modified an Escherichia coli vector expressing 66‐kDa HIV‐1 polymerase (p66) in order that it simultaneously expresses this and therefore the pol ‐coded protease. the dual expression cassette yields high quantities of both polymerase and protease; however, under these conditions, 50% of the over‐expressed p66 polymerase is processed, leading to accumulation of huge quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse‐transcriptase‐coding sequence (His‐p66) permits an easy , rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His‐p66 and His‐p66/His‐p51 polymerase exhibit both polymerase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

A water insoluble derivative of a papain inhibitor was prepared by covalently linking Gly‐Gly‐Tyr(Bzl)‐Arg to an agarose resin. The immobilized inhibitor binds active papain specifically. When papain prepared by the tactic of Kimmel and Smith was activated and applied to a column of the immobilized inhibitor at moderate ionic strength (20 mM EDTA, pH 4.3), about 50% of the entire protein wasn't bound and was found to be catalytically inactive. The bound enzyme was released with water . It contained one mole of SH per mole of protein. When assayed with α‐N ‐benzoyl‐l ‐arginine ethyl ester (25°, pH 6.0) the purified enzyme had a k cat of 28.5 sec‐1 and a K m of 18 mM. the precise activity of the purified papain was about twice that of the first preparation and therefore the K m was unchanged. The enzyme, inactivated by an equimolar amount of mercury chloride might be fully activated even after prolonged storage.

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