A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 5' portion integrates the PCR products into the homologous region of the vector. For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.

Technological advances in the field of mass spectrometry (MS) are providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion-spray) MS for the biochemical characterization of recombinant human catechol 0-methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352 rt 2Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25 775 ? 4Da.To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C-terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C-terminal extension resulted from ribosomal frameshift at the codon of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5Da) is in good agreement with our experimental result.The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S-adenosyl-L-methionine (AdoMet) and MgC12, was also studied. 5-Iodoacetamido fluorescein (5-IAF) was used as thiol-modifying reagent. Under the conditions used, 5-IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl, partially protected it from inactivation. The 5-IAF-labeled tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5-IAF. Incorporation of 5-IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl, markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.Over the last decade, electrospray and ion-spray (pneumatically assisted electrospray) mass spectrometry (MS) have found several applications in the study of protein structure (Biemann, 1990;Aebersold, 1993;Mann et al., 1993). MS efficiently complements the classical techniques of protein and peptide analysis such as N-terminal amino acid sequencing and amino acid analysis. The applications of MS range from the precise determination of the molecular mass Enzyme. Catechol 0-methyltransferase (EC 2.1.1.6).for polypeptides of >lo5 Da, and the characterization of chemical modifications occurring either as a post-translational event or during protein handlin...