44 ISSN 0003-6838, Applied Biochemistry and Microbiology, 2020, Vol. 56, No. 1, pp. 44–50. © Pleiades Publishing, Inc., 2020. Russian Text © The Author(s), 2020, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2020, Vol. 56, No. 1, pp. 52–59. The Screening of Refolding Conditions and Obtainment of the Recombinant Antistaphylococcal Endolysin LysK CA in Active Form from E. coli Inclusion Bodies A. V. Žydziecki a, *, S. G. Golenchenko a , U. A. Prakulevich a , and M. V. Sholukh a a Belarusian State University, Biology Department, Minsk, 220030 Belarus *e-mail: Zhydzetski@gmail.com Received April 17, 2019; revised July 29, 2019; accepted August 30, 2019 Abstract—Step-by-step screening of the main characteristics of refolding buffer is proposed to obtain recom- binant antistaphylococcal endolysin LysK containing two catalytic domains, CHAP and amidase-2, in active form from E. coli inclusion bodies. The optimal pH, temperature, redox potential of the refolding buffer, as well as the optimal protein concentration and type of antiaggregation compound were determined. The compo- sition of a renaturation system of antistaphylococcal endolysin containing 20 mM phosphate buffer, pH 7.4, 0.4 M sucrose, and 2.5 mM DTT at 10°C with dilution to a final concentration of ~150 μg/mL was found to be optimal. The refolding yield after scaling was about 29.5 ± 6.7%, which produced 16 ± 2.3 mg of the target protein from 2.25 g of washed inclusion bodies with a specific enzyme activity of 1.8 ± 0.73 × 10 3 U/mg. Keywords: recombinant endolysin LysK CA , protein refolding, inclusion bodies, screening of renaturation con- ditions DOI: 10.1134/S0003683820010160 INTRODUCTION The technology of recombinant DNA molecules has opened a new era in the production of various kinds of proteins, both for structural and biochemical studies and for industrial and commercial purposes [1]. Expression in transformed cells makes it possible to obtain rare, valuable, expensive proteins that are diffi- cult to obtain by traditional methods [2, 3]. In partic- ular, a number of therapeutic proteins that have great potential for use in medicine and veterinary medicine to replace standard treatment methods can be obtained with this method [4, 5]. One of these proteins is endolysin of phage K (LysK), a peptidoglycan hydrolase involved in the lysis of gram-positive bacterial cells of the Staphylococcus genus that is expressed in the terminal cycle of the release of mature phage particles from host cells [6–8]. The endolysin LysK consists of three domains, two catalytic domains (CHAP and amidase-2) and one structural (SH3b); truncated variants of this protein containing the CHAP domain also possess activity [9, 10]. The high antistaphylococcal effectiveness of LysK and its derivatives towards the Staphylococcus genus makes it a promising candidate as a new antimicrobial agent in medicine and veterinary medicine, especially with respect to antibiotic resistant strains. It should be noted that this protein has several advantages over standard antistaphylococcal drugs. The main advan- tages are the lack of the development of resistance, the manifestation of high species specificity, the absence of effects on normal microflora, and the safety of its use in the food industry [8, 11]. However, certain dif- ficulties in the study and further use of LysK and its forms are associated with their isolation in limited quantities. The research on this protein is based in most cases on its preparation in producer strains in soluble form with further purification on affinity sor- bents [7, 8, 12, 13]. However, with this technology, it is possible to obtain a small amount of protein for research purposes. In particular, recent studies obtained only 2–10 mg of CHAP K and about 12 mg of LysK CA from 1 L of fermentable culture [13–15]. At the same time, the potential applications in veterinary medicine, medicine, and other sectors of the national economy will require large quantities and, accord- ingly, cheaper and more effective methods for the pro- tein production [11]. A sufficient amount of the target protein can be prepared from inclusion bodies (IBs), dense, highly hydrated formations consisting mostly of inactive, aggregated molecules of the target pro- tein [16]. Although a number of additional difficulties arise in this case, the main issue is the activation of the target protein molecules associated with the refolding process. The effectiveness of the renaturation of recombinant proteins depends on the competition between the correct folding and aggregation [17, 18]. The dominant process is determined by the composi-