Codon optimization for enhanced Escherichia coli expression of human S100A11 and S100A1 proteins
| Autor: | Gary S. Shaw, Nicole M. Marlatt, Donald E. Spratt |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Molecular Sequence Data
Biology medicine.disease_cause Biochemistry Mass Spectrometry law.invention law S100A1 medicine Escherichia coli Coding region Humans Protein Isoforms Amino Acid Sequence Cloning Molecular EF Hand Motifs Codon Gene Nuclear Magnetic Resonance Biomolecular Conserved Sequence Cloning Base Sequence Nitrogen Isotopes EF hand Oligonucleotide S100 Proteins Nuclear magnetic resonance spectroscopy Codon optimization Molecular biology S100A11 Recombinant DNA Electrophoresis Polyacrylamide Gel Sequence Alignment Biotechnology |
| Zdroj: | Biochemistry Publications |
| ISSN: | 1096-0279 |
| Popis: | The cloning, expression and purification for the recombinant full-length human proteins S100A11 and human S100A1 is described. The genes were synthesized by overlapping complementary single-stranded oligonucleotides of various lengths. The coding sequence for both genes were codon optimized by selecting only the most preferential codons according to the Escherichia coli bias. In order to assemble the various oligonucleotides into the correct full-length genes, a unique one-step PCR procedure was implemented. The expression and purification procedures were also optimized for each protein. A single phenyl-Sepharose column was sufficient for the purification of human S100A11 whereas HiTrap Q anion exchange followed by phenyl-Sepharose columns were required for the purification of S100A1. By optimizing the S100A1 and S100A11 gene, expression and purification protocols, more than 45 and 150 mg, respectively of the purified human proteins were obtained per litre of media. Protein identity was verified by both SDS–PAGE and mass spectrometry (MS) and further characterized by NMR spectroscopy. These results have established an efficient method for the expression and purification of large quantities of human S100A1 and S100A11 proteins for biophysical characterization. |
| Databáze: | OpenAIRE |
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