Abstract
BackgroundRecombinant human Fibroblast growth factor 21 (rhFGF21) is an endocrine hormone that has profound effects on treatment of metabolic diseases. However, rhFGF21 is prone to form inclusion body when expressed in bacteria, which results in, the downstream process of purification of bioactive rhFGF21 is time-consuming and labor intensive. The aim of this work is to explore a new method for improving the soluble expression and secretion level of rhFGF21 in B. subtilis.ResultsA codon optimized rhFGF21 gene was expressed under the control of a strong inducible promoter PmalA in B. subtilis. A mini-cistron cassette (from gsiB) was located upstream of rhFGF21 in expression vector (pMATEFc5), which could reduce the locally stabilized mRNA secondary structure of transcripts and enhance the efficiency of translation initiation. Then various chaperones were further overexpressed to improve the expression efficiency of rhFGF21. Results showed that overexpression of the chaperone DnaK contributed to the increase of solubility of rhFGF21. Moreover, an extracellular proteases deficient strain B. subtilis Kno6cf was used to accumulate the secreted rhFGF21 solidly. In addition, eleven signal peptides from B. subtilis were evaluated and the SPdacB appeared the highest secretion yield of rhFGF21 in B. subtilis. Finally, the combinatorial optimized strain achieved an about ninefold increase of the soluble rhFGF21 production after 24 h of flask fermentation in comparison with the initial production strain.ConclusionThis work provided a comprehensive strategy for secretory expressing the heterologous protein rhFGF21 in B. subtilis. To our knowledge, this is the first report of the highly efficient production of rhFGF21 in B. subtilis and this approach may provide some suggestions for heterologous proteins production in B. subtilis.
Highlights
Recombinant human Fibroblast growth factor 21 is an endocrine hormone that has profound effects on treatment of metabolic diseases
Strategies designed for improving Recombinant human Fibroblast growth factor 21 (rhFGF21) protein expression in B. subtilis In order to secretory express the target protein rhFGF21 in B. subtilis, different strategies at the level of transcription, translation, protein folding/degrading, translocation and secretion process were performed to address potential bottlenecks in eukaryotic protein expression (Fig. 1), which includes selection of strong promoters, introduction of a mini-cistron cassette to enhance translational initiation, overexpression of different chaperones, and selection of a optimum signal peptide
In an effort to improve the soluble expression of the rhFGF21 protein in B. subtilis, we evaluated the overexpression of individual chaperones using 11 genes or gene operons involved in protein folding or translocation, and found that the overexpression of the dnak operon markedly improved the production of rhFGF21 (Fig. 4)
Summary
Recombinant human Fibroblast growth factor 21 (rhFGF21) is an endocrine hormone that has profound effects on treatment of metabolic diseases. The aim of this work is to explore a new method for improving the soluble expression and secretion level of rhFGF21 in B. subtilis. The full-length hFGF21 consists of 209 amino acids with a signal peptide of 28 amino acids at the N-terminus, splicing a mature FGF21 polypeptide of 181 amino acids. As a novel endocrine hormone, FGF21 has profound effects on the regulation of metabolic parameters such as glucose and lipid homeostasis, and represents a promising potential therapeutic target in type 2 diabetes (T2D) and obesity [3]. Recombinant human fibroblast growth factor 21 (rhFGF21) has been a focus of investigation as a new drug candidate for metabolic diseases [3]
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