Improving the Expression of Human Granulocyte Colony Stimulating Factor in Escherichia coli by Reducing the GC-content and Increasing mRNA Folding Free Energy at 5’-Terminal End

Purpose : Strategy for improving the production of biopharmaceutical protein continues to develop due to increasing market demand. Human granulocyte colony stimulating factor (hGCSF) is one of biopharmaceutical proteins that has many applications, and easily produced in Escherichia coli expression system. Previous studies reported that codon usage, rare codon, mRNA folding and GC-content at 5’-terminal end were crucial for protein production in E. coli. In the present study, the effect of reducing the GC-content and increasing the mRNA folding free energy at the 5’-terminal end on the expression level of hG-CSF proteins was investigated. Methods: Synonymous codon substitutions were performed to generate mutant variants of open reading frame (ORF) with lower GC-content at 5’-terminal ends. Oligoanalyzer tool was used to calculate the GC content of eight codons sequence after ATG. Whereas, mRNA folding free energy was predicted using KineFold and RNAfold tools. The template DNA was amplified using three variant forward primers and one same reverse primer. Those DNA fragments were individually cloned into pJexpress414 expression vector and were confirmed using restriction and DNA sequencing analyses. The confirmed constructs were transformed into E. coli NiCo21(DE3) host cells and the recombinant protein was expressed using IPTG-induction. Total protein obtained were characterized using SDS-PAGE, Western blot and ImageJ software analyses. Results: The result showed that the mutant variant with lower GC-content and higher mRNA folding free energy near the translation initiation region (TIR) could produce a higher amount of hG-CSF proteins compared to the original gene sequence. Conclusion: This study emphasized the important role of the nucleotide composition immediately downstream the start codon to achieve high-yield protein product on heterologous expression in E. coli.

Structural Characteristics in the γ Chain Variants Associated with Fibrinogen Storage Disease Suggest the Underlying Pathogenic Mechanism

Particular fibrinogen γ chain mutations occurring in the γ-module induce changes that hamper γ-γ dimerization and provoke intracellular aggregation of the mutant fibrinogen, defective export and plasma deficiency. The hepatic storage predisposes to the development of liver disease. This condition has been termed hereditary hypofibrinogenemia with hepatic storage (HHHS). So far, seven of such mutations in the fibrinogen γ chain have been detected. We are reporting on an additional mutation occurring in a 3.5-year-old Turkish child undergoing a needle liver biopsy because of the concomitance of transaminase elevation of unknown origin and low plasma fibrinogen level. The liver biopsy showed an intra-hepatocytic storage of fibrinogen. The molecular analysis of the three fibrinogen genes revealed a mutation (Fibrinogen Trabzon Thr371Ile) at exon 9 of the γ chain in the child and his father, while the mother and the brother were normal. Fibrinogen Trabzon represents a new fibrinogen γ chain mutation fulfilling the criteria for HHHS. Its occurrence in a Turkish child confirms that HHHS can present in early childhood and provides relevant epidemiological information on the worldwide distribution of the fibrinogen γ chain mutations causing this disease. By analyzing fibrinogen crystal structures and calculating the folding free energy change (ΔΔG) to infer how the variants can affect the conformation and function, we propose a mechanism for the intracellular aggregation of Fibrinogen Trabzon and other γ-module mutations causing HHHS.