Designing Signal Peptides for Efficient Periplasmic Expression of Human Growth Hormone in Escherichia coli

Abstract

The secretion efficiency of a protein in a Sec-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, namely, two synthetic Sec-type and three alpha-amylase-derived signal peptides, were compared for periplasmic expression of the human growth hormone (hGH) in. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for hGH secretion in , differ in their N-terminal positively charged residues and hydrophobic region lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing a specific signal peptide by using a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.

Enhanced Extracellular Production of IsPETase in Escherichia coli via Engineering of the pelB Signal Peptide

“…61 For pelB WT and variants, mRNA sequence including the ribosome binding site (RBS), translation start codon, signal sequence, and six nucleotides after the end of the signal peptide-coding region (104 bases total) was analyzed to predict its possible secondary structure and the folding free energy (ΔG). 51,52,62 ■ RESULTS AND DISCUSSION Construction of the Secretory Expression Vector and the Expression of Extracellular PETase in E. coli BL21-Gold (DE3). The gene fragment encoding PETase was cloned into the expression vector pET22b, which contains the signal peptide, pelB, upstream of the multiple cloning site.…”

JetGene—Online Database and Toolkit for an Analysis of Regulatory Regions or Nucleotide Contexts at Differently Translated Plants Transcripts

“…This module makes it possible to display an amino acid that is located on sequence in the position 1-10 both from C-terminus and from N-terminus. It can be helpful for an analysis and designing of signal peptides [7] and for applying the N-end rule. According to this rule the second N-terminal amino acid of a protein determines its half-life [8].…”

Efficient Interleukin-21 Production by Optimization of Codon and Signal Peptide in Chinese Hamster Ovarian Cells

“…Five different signal peptides were identified by literature search and fused with the codon-optimized mature IL-21 [16][17][18][19]. The origin and amino acid sequence of signal peptides are shown in Table 1.…”

“…A number of parameters, including codon usage, mRNA stability and the GC content, and RNA instability motifs and splicing sites, are considered for gene optimization to improve transcription, translation, and folding of recombinant protein [15,16]. The selection of proper signal peptides is also critical for establishing a manufacturing process for protein production to improve the correct processing and secretion of recombinant proteins through the transport of the translated proteins into the endoplasmic reticulum [17,18]. Recent studies have demonstrated the effectiveness of different signal peptides on the production of recombinant protein in CHO cells [19][20][21][22].…”