Vector-swapping strategy with an exonuclease in cloning and high expression of small PCR products.

The need for designing new peptides of biological, pharmacological or clinical importance is increasing. To fulfill this need, a detailed knowledge of the structure-function relationship of known bioactive proteins is a prerequisite, and to obtain such information, we face challenges of inserting and deleting specific sequences into or from known proteins and expressing these modified proteins in cells. Polymerase chain reaction (PCR) (4) is essential for accomplishing the former procedure, and it is also a useful method for localizing the target function in short sequences, such as oligopeptides. Such studies require handling of small PCR products coding for the target areas of the proteins. These products are usually separated and isolated on polyacrylamide or agarose gels prior to expression in cells. However, this procedure is laborious and time-consuming and often results in a low recovery of the products. We can solve this problem by devising a new method to clone and express small DNAs that does not require any separation and isolation steps. The cloning and expression strategy are schematically outlined in Figure 1. Two commonly used vectors were selected for cloning and expression, the pCRII vector for cloning (donor) and the pMAL-c2 vector for expression (acceptor). pCRII and pMAL-c2 vectors were obtained from Invitrogen (Carlsbad, CA, USA) and New England Biolabs (Beverly, MA, USA), respectively. These vectors share an ampicillin-resistance (ampr) gene. This shared gene in the cloning vector was selectively destructed after pCRII cloning. In this cloning step, the PCR products are amplified using PCR primers flanked with BamHI recognition sequence. This flanking sequence was designed so as to adjust the reading frame of the PCR products to that of the malE gene in the pMAL-c2 vector. Another technique was devised for direct PCR cloning and expression of a concerned protein. In this procedure, PCR primers were designed to incorporate unique restriction endonuclease sites at their 5′ ends without altering the translation reading frame of the protein (2). PCR products were then cleaved by the endonuclease to create sticky ends, followed by a direct ligation to an expression vector that had been cut with the same endonuclease. However, many restriction endonuclease sites fail to be cleaved when their recognition sequences are located within a few base pairs of the end of a DNA fragment (2). One way to overcome this difficulty is to clone the PCR product as a bluntended fragment before its expression. However, in this case, an additional procedure is required in which a 3′ overhang of the PCR products should be enzymatically removed using an enzyme such as Klenow or T4 DNA polymerase (6). The usefulness of the innovated method was tested for the small PCR products (207 and 285 bp) derived from human fibronectin cDNA (1). In brief, the DNA fragments were amplified by PCR by using cDNA clones encoding human fibronectin, Taq DNA poly-