Cloning, expression, purification and amyloid degradation study of recombinant serrapeptase from Serratia marcescens

Background Serrapeptase (SP) is a proteolytic enzyme which plays a prominent role in the degradation of dead tissue. It can also use as a therapeutic enzyme for the treatment of Insulin amyloids. It has reported that the SP protein has the fibrinolytic activity, but the activities towards insulin amyloid degradation not reported. Our study has focused the cloning and expressing SP gene and studying its cytotoxic reactivity studies and amyloids degradation. Results SP gene was cloned in pET-28 a (+), and the enzyme was expressed using E.coli BL 21 (DE3) strain. The clone was confirmed using colony PCR, sequencing and restriction digestion. Overexpression of enzyme and purification performed using IPTG and Ni-NTA column. The molecular weight of serrapeptase protein was determined using SDS-PAGE, which found to be 52 kDa. The purified enzyme showed a zone of clearance on casein plate, slight cytotoxic reactivity to PC-12 cells after 24 h contact and 25% insulin amyloid degradation confirmed after 24 h of incubation. Conclusion This study shows that cloning and the expression of SP will reduce the time and cost of production of SP from wild type strain which can be used for the treatment of amyloid-related diseases.

[1]  K. N. Rajnish,et al.  Enhancement of Serrapeptase Hyper Producing Mutant by Combined Chemical and UV Mutagenesis and its Potential for Fibrinolytic Activity , 2020 .

[2]  K. N. Rajnish,et al.  Enzyme therapy: a forerunner in catalyzing a healthy society? , 2020, Expert opinion on biological therapy.

[3]  S. Gopinath,et al.  Production, purification, characterization, immobilization, and application of Serrapeptase: a review , 2017, Frontiers in Biology.

[4]  U. Luthra,et al.  Analytical techniques for serratiopeptidase: A review , 2017, Journal of pharmaceutical analysis.

[5]  Manoj Kumar,et al.  Microbial enzymes: industrial progress in 21st century , 2016, 3 Biotech.

[6]  S. Bhagwat,et al.  Purification of serratiopeptidase from Serratia marcescens NRRL B 23112 using ultrasound assisted three phase partitioning. , 2016, Ultrasonics sonochemistry.

[7]  M. R. Nilsson.  Insulin amyloid at injection sites of patients with diabetes , 2016, Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis.

[8]  V. Suryanarayana,et al.  IN SILICO ANALYSIS OF INHIBITOR AND SUBSTRATE BINDING SITE OF SERRAPEPTIDASE FROM SERRATIA MARCESCENS MTCC 8708 , 2016 .

[9]  P. Pucci,et al.  Serratiopeptidase: a well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm , 2015, BMC Microbiology.

[10]  M. Benson,et al.  Nomenclature 2014: Amyloid fibril proteins and clinical classification of the amyloidosis , 2014, Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis.

[11]  M. Dimopoulos,et al.  A European collaborative study of treatment outcomes in 346 patients with cardiac stage III AL amyloidosis. , 2013, Blood.

[12]  Adriano Brandelli,et al.  Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3 , 2012 .

[13]  A. Tariq,et al.  Purification and Characterization of 56 KDa cold active Protease from Serratia marcescens , 2011 .

[14]  D. Seldin,et al.  Amyloidosis: pathogenesis and new therapeutic options. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[15]  Kuan-Nan Liu,et al.  Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants. , 2010, Biochimica et biophysica acta.

[16]  F. Sharp,et al.  Genome response to tissue plasminogen activator in experimental ischemic stroke , 2010, BMC Genomics.

[17]  An-Na Li,et al.  Cloning, expression and characterization of the serine protease gene from Chaetomium thermophilum , 2009, Journal of applied microbiology.

[18]  U. Baumann,et al.  The conserved methionine residue of the metzincins: a site-directed mutagenesis study. , 2001, Journal of molecular biology.

[19]  S. C. Braunagel,et al.  The metalloprotease gene ofSerratia marcescens strain SM6 , 1990, Molecular and General Genetics MGG.

[20]  H. Matsubara,et al.  Cloning and sequencing of Serratia protease gene. , 1986, Nucleic acids research.

[21]  A. Girigoswami,et al.  In vitro and in vivo insulin amyloid degradation mediated by Serratiopeptidase. , 2017, Materials science & engineering. C, Materials for biological applications.

[22]  R. Puvanakrishnan,et al.  Enzyme Therapy: Current Perspectives. , 2016, Indian journal of experimental biology.

[23]  V. Suryanarayana,et al.  Characterization of a Gene Encoding Serrapeptidase from Serratia Marcescens Strain (SRM) MTCC 8708, a Plant Isolate , 2015 .

[24]  F. Fanglian He,et al.  Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method , 2011 .

[25]  G. Klein,et al.  Short-Term Treatment of Painful Osteoarthritis of the Knee with Oral Enzymes , 2000 .