pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli.
The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.
Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
Zelong
Z
Ma
Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
Dan
D
Luo
State Key Laboratory for Gene and Cell Therapy, Sichuan University, Chengdu, China.
Anliang
A
Huang
Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China.
Yang
Y
Xu
Clover Biopharmaceuticals, Chengdu, China; State Key Laboratory for Gene and Cell Therapy, Sichuan University, Chengdu, China.
Yunxiao
Y
Wang
State Key Laboratory for Gene and Cell Therapy, Sichuan University, Chengdu, China.
Yuquan
Y
Wei
Center for Growth, Metabolism and Aging, School of Life Sciences, Sichuan University, Chengdu 610064, China; Clover Biopharmaceuticals, Chengdu, China; State Key Laboratory for Gene and Cell Therapy, Sichuan University, Chengdu, China; GenHunter Corporation, 624 Grassmere Park, Nashville, TN 37211, USA. Electronic address: liangpeng@scu.edu.cn.
Peng
P
Liang
D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D017931:DNA Primers; C554482:KLLN protein, human; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins
Bacterial expression; Cloning vector; His-tag; Killin; PCR cloning; Positive-selection