ABSTRACT
Enterokinase is the protease of choice for N-terminal fusions, since it specifically recognizes a five amino-acid polypeptide (D-D-D-D-K-X1) and cleaves at the carboxyl site of lysine. Sporadic cleavage at otherresidues was observed to occur at low levels, dependingon the conformation of the protein substrate [1]. Biochemical analyses haveshown that the cleavage efficiency depends on the aminoacid residue X1 downstream of the D4K recognition site [2]. Recombinant hybrids containing apolypeptide fusion partner, termed affinity tag, to facilitatethe purification of the target polypeptides are widelyused. Many different proteins, domains, or peptides canbe fused with the target protein. The advantages of usingfusion proteins to facilitate purification and detection ofrecombinant proteins are well-recognized [3].All tags, whether large or small, have the potential to interfere with the biological activity of a protein, impede its crystallization, or otherwise influence its behavior.Consequently, it is usually desirable to remove the tag [4]. Therefore various proteases like factor Xa, recombinant bovine, enterokinase, etc., were used for tag separation.Protease activity is also regulated by metal ion cofactors that reversibly bind to proteases and affect their final activity, often in an allosteric manner [5]. Most of the first row transition metals (the only exceptions being scandium, titanium, and perhaps chromium) as well asmolybdenum; tungsten and magnesium are known to function as cofactors in enzymatic catalysis. Typically, these metal-ionic cofactors are bound to the enzyme viacoordination of amino acid side-chains. Enzymes with more looselybound metal ion cofactors, commonly called metalactivatedenzymes, require the presence of the appropriatemetal ion in the buffer in order to observe maximalcatalytic activity [6]. The metal cofactors, Mg2+ and Mn2+, appear to stabilize two distinct conformational states of the enzyme which differ in response to varying substrate and effector concentrations [7]. The aim of the present investigation is to study the effect of cofactor Magnesium ion (Mg2+) ion on the activity of recombinant bovine enterokinase.
