RESUMEN
The manipulation of recombinant DNA has been an integral step in molecular biology to date. A number of strategies have been developed over the years, as traditional cloning methods are time consuming, have high backgrounds and low efficiency and are often limited by the number of suitable restriction sites available. Here, we constructed a series of new positive-selection-based cloning vectors that overcome most of the above mentioned drawbacks and can be applied in both eukaryotic and prokaryotic systems. This strategy is based on the extreme toxicity of DpnI in wild-type E. coli and the inactivation of this lethality by the introduction of target gene within multiple cloning sites. There are no rapid approaches for identifying soluble proteins for high-throughput screening. In this study, we combined this highly efficient cloning strategy with rapid identification of soluble proteins to construct vectors with multiple fusion tags, such as MBP, GST, CBD, NusA, and Sumo, to generate enzymes with potential diagnostic, industrial or therapeutic applications. Thus, this versatile positive-selection-based technology is appropriate for routine cloning, DNA library construction, and high-throughput screening for the expression of proteins of interest.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Clonación Molecular , ADN/análisis , Escherichia coli/genética , Perfilación de la Expresión Génica , Vectores Genéticos , Células HEK293 , Humanos , Metilación , Proteínas Recombinantes de Fusión/metabolismo , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/genéticaRESUMEN
OBJECTIVE: To fabricate an active and stable enzyme through covalent immobilization, a Ubl-specific protease (Ulp1) was used to cleave small ubiquitin-like modifier (SUMO) fusion proteins. RESULTS: We immobilized Ulp1 on N-hydroxysuccinimide (NHS)-activated Sepharose with a coupling efficiency of 1.7 mg/ml. The immobilized Ulp1 maintains 95% substrate-cleavage ability and significantly enhances pH and thermal stability, especially can withstand pH of 10.5. Besides resistance against some small molecules, the immobilized Ulp1 can tolerate 15% (v/v) DMSO and 20% (v/v) ethanol. It can be reused for more than 15 batch reactions with 90% activity retention. This provides a fast purification system to quickly obtain cleaved recombinant proteins with 95% purity from cell lysates with the application of immobilized Ulp1. CONCLUSIONS: Ulp1 used in immobilization form is a potentially useful tool for cleavage of SUMO-tagged proteins and may reduce time and cost of protein purification.