A New Generation of Cold-shock Vector Derived from pCold I (pCold I-LZ) for Expression of Recombinant Peptides and Proteins

Abstract

Background:

Finding shows that there were several problems in the treatment of Helicobacter pylori infection such as the emergence of resistance to the antibiotics, the risk of recrudescence, and the high cost of treatment. The ineffectiveness of conventional treatment mechanisms against cancer cells reveals the importance of peptides as a novel therapeutic approach. However, the short length of peptides was a restriction factor to realise this approach. As a result, one or more amino acid residues were added during the cloning process that leads to the loss of the original peptide folding. The aim of study was to change the structure of the pCold I vector using site-directed mutagenesis in order to do the directional cloning of each target gene and express/purify the small peptides and native proteins without any additional N-terminus amino acids.

Materials and Methods:

Site-directed mutagenesis was performed by plasmid amplification in two individual PCR reactions. Two PCR products were mixed and denatured to separate the nascent strands of plasmid DNA from the parental ones. Slow cooling conditions were used to allow reannealing of PCR products. The products were digested with DpnI that digests methylated parental strands, and then transformed into E. coli DH5α. The mutant plasmid was identified by digestion with NdeI and PceI and sequencing.

Results:

The mutant plasmids were not digested by NdeI. The presence of mutation was also confirmed by sequencing.  In mutant vector the cut sites of both the first restriction enzyme at the multiple cloning site (at the nucleotide level) and factor Xa (at the amino acid level) became the same.

Conclusion:

The modified vector can be widely used for a fast and more convenient cloning and expression of the native proteins and short peptides, including anti-H. pylori peptides and anti-angiogenesis, gastric anti-CSCs and anti-metastatic peptides.