title: Comparative analysis of the effectiveness of C-terminal cleavage intein-based constructs in producing a recombinant analog of anophelin, an anticoagulant from Anopheles albimanus.
abstract: Production of small recombinant peptides by expressing them as fusion proteins, with subsequent proteolytic or chemical cleavage of the latter, is a widespread approach in modern biotechnology. An alternative method is to produce such peptides as self-cleaving fusion proteins with inteins. To date, only a small proportion of known inteins have been used for this purpose, and analysis of other inteins for the ability to cleave off the target polypeptide can significantly expand the range of intein-based transgenic constructs available to researchers. Most interesting in practical terms are С-terminal cleavage constructs for producing target polypeptides without an N-terminal methionine residue. We prepared two new such constructs with mini-inteins GyrA from Mycobacterium xenopi and RIR1 from Methanobacterium thermoautotrophicum. Together with the previous construct based on the artificial mini-intein derived from Synechocystis sp. DnaB intein, they were used to produce a recombinant analog of anophelin, the naturally occurring thrombin inhibitor from the mosquito Anopheles albimanus. The effectiveness of the constructs with Ssp DnaB and Mth RIR1 proved to be relatively low because of spontaneous fusion protein cleavage during the producer strain culturing in the former case and a low degree of its cleavage upon purification in the latter case. The most effective Mxe GyrA construct was used to develop a semipreparative procedure for producing recombinant anophelin, with its yield reaching 91 ± 2 mg protein per liter of culture medium. As determined by an amidolytic assay, the antithrombin activity and K i of recombinant anophelin were 3362.8 ATU/mg and 87 ± 3 рМ, respectively.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St., Moscow, Russia, 117997, esipov@ibch.ru.
authors.forename: Roman S
authors.initials: RS
authors.lastname: Esipov
authors.forename: Maria A
authors.initials: MA
authors.lastname: Kostromina
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000852:Anopheles / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000925:Anticoagulants / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D047668:Inteins; D007700:Kinetics; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry
chemical_list: D000925:Anticoagulants; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides; C121275:anophelin protein, Anopheles albimanus; D013917:Thrombin