Designing and constructing recombinant expression cassettes for production and secretion of recombinant proteins in the hairy root

Abstract

Objective The use of plant expression systems to produce recombinant proteins have advantages such as different methods for transformation and culture, occurrence of appropriate post-translational modifications and no risk of contamination with animal and microbial pathogens. However, low productivity in these expression systems have always been a major challenge. Secretion-based expression systems, such as Hairy Root Cultures (HRCs), is a convenient way to continuously produce active ingredients without the need for plant tissue degradation, which will facilitate the purification process and significantly reduce related costs. This study aimed to designing and constructing vectors carrying NtREL1 root specific promoter in combination with patatin, pectin methyl esterase and calreticulin signal peptides at the same time in order to increase the specific expression of recombinant proteins in hairy root culture and their subsequent secretion into hydroponic medium. Materials and methods The nucleotide sequence of NtREL1 root specific promoter, patatin, pectin methyl esterase and calreticulin signal peptides were retrieved from NCBI database. To identify cis acting regulatory elements involved in specific expression, the NtREL1 sequence was investigated by PLACE and PLANT CARE softwares. Bioinformatics studies were performed to design pBI121 expression vector with NtREL1 promoter in combination with signal peptides and appropriate restriction sites. After receiving the synthetic fragments in pUC57 vector, the fragments were digested by restriction endonuclease and sub cloned in pBI121 expression vector. Results Examination of the NtREL1 promoter showed that this promoter is rich in AT nucleotides. These sequences can improve the recognition process by transcription systems and increase gene expression due to their ease of conversion from double-stranded to single-stranded form. The accuracy of NtREL1 promoter sub cloning upstream of patatin, pectin methyl esterase and calreticulin signal peptides in pBI121 expression vector were confirmed using colony PCR, digestion reactions and sequencing. Conclusions Since pBI121-based expression vectors are more efficient than other expression vectors and widely used in genetic transformation and expression of recombinant proteins in plants, this recombinant vectors can be applied effectively for recombinant protein production and its secretion in expression systems like hairy root culture.

Keywords

• expression vector
• root specific promoter
• signal peptide