title: Optimization of expression of untagged and histidine-tagged human recombinant thrombin precursors in Escherichia coli.
abstract: The present study is focused on preparation of proper Escherichia coli expression system to ensure high yields of various modified precursors of human recombinant thrombin, a potential biopharmaceutical reagent. Two thrombin precursors, the smallest single-chain α-thrombin precursor prethrombin-2 and its shortened form prethrombin-2∆13, and their His-tagged forms were used. In order to determine the effect of the different lengths and amino acid compositions of affinity His-tag on the target protein expression level, a variety of the His-tag sequences were used. We found out that the protein expression efficiency was closely related to the codons used for encoding of amino acids of fusion histidine tag. Optimization of culture medium composition is another way to increase yield of the target protein. Suitable medium composition can ensure cell growth to high densities which is related to total yield of expressed protein. In this study, a new optimized complex medium for batch fermentation was developed. Addition of nutrients like a yeast extract and enzymatic casein hydrolysate to the defined medium components had a positive impact on protein expression, where relatively high expression level of the target protein from total amount of cellular proteins was achieved. Further, we have focused on trace element solution composition, and the optimized nickel and selenium concentrations were determined. Our results show that the composition of essential trace metal solution has a major impact not only on expression level, but it can also affect cell growth rate.
authors.affiliation: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina, 842 15, Bratislava 4, Slovak Republic, osadska@fns.uniba.sk.
authors.forename: Michaela
authors.initials: M
authors.lastname: Osadská
authors.forename: Hana
authors.initials: H
authors.lastname: Boňková
authors.forename: Ján
authors.initials: J
authors.lastname: Krahulec
authors.forename: Stanislav
authors.initials: S
authors.lastname: Stuchlík
authors.forename: Ján
authors.initials: J
authors.lastname: Turňa
mesh_terms: D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011516:Prothrombin / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D003470:Culture Media; D011993:Recombinant Fusion Proteins; D011516:Prothrombin; C012007:prethrombins