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.

Screening of the Promising Direct Thrombin Inhibitors from Haematophagous Organisms. Part I: Recombinant Analogues and Their Antithrombotic Activity In Vitro

“…Expression constructs were prepared using the plasmid vectors pAL2-T (Evrogen, Moscow, Russia), pTWIN1 (New England Biolabs, Ipswich, MA, USA), and pERIG [ 37 ], Encyclo DNA polymerase (Evrogen), restriction endonucleases NdeI, BamHI, and LguI, and T4 DNA ligase (Thermo Fisher Scientific, Burlington, ON, Canada). The proteins were chromatographically purified on XK 16/20 (lab-scale process), HiScale 50/40, and HiScale 50/20 columns (semi-prep scale process) with Q Sepharose XL and Q Sepharose HP resins (Cytiva, Danaher Corporation, Washington, DC, USA), a Kromasil 300-10-C18 RP-HPLC column (Kromasil, Nouryon, Bohus, Sweden), and BPG 140/750 column with Bio-Gel P-2 media (Bio-Rad Laboratories, Hercules, CA, USA).…”

“…Thromborel-S, Pathromtin SL, and Test Thrombin reagents from Siemens Healthcare Diagnostic (Erlangen, Germany) were used in coagulation tests with human plasma. Recombinant hirudin-1 and anophelin were obtained according to the protocol published previously [ 36 , 37 ]. Other reagents used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Panreac (Barcelona, Spain).…”

“…The haemadin gene was subcloned into the original expression vector pERIG atLguI and BamHI restriction sites [ 37 ]. The variegin gene was subcloned into the expression vector pTWIN1 atNdeI and SapI restriction sites.…”

“…In our previous papers, we have reported the production of the recombinant hirudin-1 and anophelin, an anticoagulant from the malaria mosquito Anopheles albimanus [ 36 , 37 ]. According to the published data, solid-phase peptide synthesis is still the only effective method to produce variegin [ 38 ].…”

Inteins as tools for tagless and traceless protein purification

“…Truncated RNAse, truncated Hpal Paramoysin, Thioredoxin Trx RNAP CM4, HβD4 Hstl Anophelin CHAP Pa‐MAP 2 Thymosin B4…”