Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon α-2b
Escherichia coli is an extensively used host for the production of recombinant proteins, making its N-terminal methionine aminopeptidase (MAP) an attractive candidate for studies on posttranslational protein processing. The present study describes the recombinant production and properties of MAP from the DH5α strain of E. coli. The soluble and active enzyme was produced in E. coli BL21 (DE3) RIL - codon plus cells under a T7 promoter system and purified by anion-exchange chromatography. It exhibited a molecular weight of 29,200.94 Da by MALDI-TOF analysis. The purified enzyme showed specific activity of 1.64 U/mg with methionylp-nitroanilide and 1.51 U/mg with synthetic tetrapeptide substrate `MGMM' in a discontinuous HPLC-based assay. In vitro studies showed the processing of up to 36% of Met-INFα-2b in 40 min. In silico studies revealed that the ES-complex formation between the enzyme and interferon has a ΔG -683.07 kJ/mol. Molecular docking results showed that the processed INFα-2b has greater binding affinity with IFNAR2 receptor as indicated by ΔG -784.53 kJ/mol, significantly lower than that of methionine containing INFα-2b (ΔG -717.63 kJ/mol). These findings emphasize the functional superiority or better efficacy of N-terminal methionine processed recombinant interferon.
___
- Aksnes H, Drazic A, Arnesen MM (2016). First things first: vital
protein marks by n-terminal acetyltransferases. Trends
Biochem Sci 41: 746-760.
- Allen WJ, Balius TE, Mukherjee S, Brozell SR, Moustakas DT, Lang
PT, Case DA, Kuntz ID, Rizzo RC (2015). DOCK 6: impact
of new features and current docking performance. J Comput
Chem 36: 1132-1156.
- Arfin SM, Bradshaw RA (1988). Cotranslational processing and
protein turnover in eukaryotic cells. Biochem 27: 7979-7984.
- Arif A, Gardner, QAA, Rashid N, Akhtar M (2015). Production of
human interferon alpha-2b in
Escherichia coli
and removal
of N-terminal methionine utilizing archaeal methionine
aminopeptidase. Biologia 70: 282-287.
- Arif A, Rashid N, Aslam F, Mahmood N, Akhtar M (2016). Biased
expression, under the control of single promoter, of human
interferon α-2b and
Escherichia coli
methionine amino
peptidase genes in
E. coli
, irrespective of their distance from
the promoter. Pak J Pharm Sci 29: 375-379.
- Ben-Bassat A, Bauer K, Chang SY, Myambo K, Boosman A, Chang S
(1987). Processing of the initiation methionine from proteins:
properties of the
Escherichia coli
methionine aminopeptidase
and its gene structure. J Bacteriol
169: 751-757.
- Bingel-Erlenmeyer R, Kohler R, Kramer G, Sandikci A, Antolic S,
Maier T, Schaffitzel C, Wiedmann B, Bukau B, Ban N (2008). A
peptide deformylase-ribosome complex reveals mechanism of
nascent chain processing. Nature 452: 108-111.
- Bradford MM (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72: 248-254.
- Calcagno S1, Klein CD (2016). N-Terminal methionine processing
by the zinc-activated
Plasmodium falciparum
methionine
aminopeptidase 1b. Appl Microbiol Biotechnol 100: 7091-
7102.
- Cantu-Bustos J, Kevin ED, Villar CD, Vargas-Cortez T, Morones-
Ramirez JR, Balderas-Renteria I, Zarate X (2016). Recombinant
protein production data after expression in the bacterium
Escherichia coli
. Data in Brief 7: 502-508.
- Chen A, Sun Y, Zhang W, Peng F, Zhan C, Liu M, Yang Y, Bai Z (2016).
Downsizing a pullulanase to a small molecule with improved
soluble expression and secretion efficiency in
Escherichia coli
.
Microb Cell Fact 15: 9.
- Chill JH, Quadt SR, Levy R, Schreiber G, Anglister J (2003). The
human type I interferon receptor: NMR structure reveals the
molecular basis of ligand binding. Structure 11: 791-802.
- De Weerd NA, Samarajiwa SA, Hertzog PJ (2007). Type I interferon
receptors: biochemistry and biological functions. J Biol Chem
282: 20053-20057.
- Dolores DNM, Ortiz A (2016). Enzyme replacement therapy for
Fabry disease. J Inborn Errors Metabol Screen 4: 1-7.
- Elleuche S, Schäfers C, Blank S, Schröder C, Antranikian G
(2015). Exploration of extremophiles for high temperature
biotechnological processes. Curr Opin Microbiol 25: 113-119.
- Giglione C, Boularot A, Meinnel T (2004). Protein N-terminal
methionine excision. Cell Mol Life Sci 61: 1455-1474.
- Goh HC, Sobota RM, Ghadessy FJ, Nirantar S (2017). Going native:
complete removal of protein purification affinity tags by
simple modification of existing tags and proteases. Prot Exp
Purification 129: 18-24.
- Gull I, Samra ZQ, Aslam MS (2013). Heterologous expression,
immunochemical and computational analysis of recombinant
human interferon alpha 2b. SpringerPlus 2: 264.
- Helgren TR, Wangtrakuldee P, Staker BL, Hagen TJ (2016). Advances
in bacterial methionine aminopeptidase inhibition. Curr Top
Med Chem 16: 397-414.
- Jia B, Jeon CO (2016). High-throughput recombinant protein
expression in
Escherichia coli
: status and future perspectives.
Open Biol
6: 160196.
- Kanodia JS, Kim Y, Tomer R, Khan Z, Chung K, Storey JD, Lu H,
Keller PJ, Shvartsman SY (2011). A computational statistics
approach for estimating the spatial range of morphogen
gradients. Development 138: 4867-4874.
- Karlberg H, Lindegren G, Mirazimi A (2010). Comparison of
antiviral activity of recombinant and natural interferons against
Crimean-Congo hemorrhagic fever virus. Open Virol J 4: 38.
- Laemmli UK (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227: 680-685.
- Lagasse HA, Levin DD, Hengel H, Golding B, Sauna ZE (2017). Fc-
fusion drugs have C1q/FcγR binding and signaling properties
that can affect their immunogenicity. J Immunol 198: 129.13.
- Liao YD, Jeng DJ, Wang CF, Wang SC, Chang ST (2004). Removal
of N-terminal methionine from recombinant proteins by
engineered
E. coli
methionine aminopeptidase. Protein Sci 13:
1802-1810.
- Lovell SC, Davis IW, Arendall WB, de Bakker PIW, Word JM,
Prisant MG, Richardson JS, Richardson GC (2002). Structure
validation by Calpha geometry: phi, psi and Cbeta deviation.
Proteins: Structure, Function & Genetics 50: 437-450.
- Lowether WT, Matthews BW (2000). Structure and function of the
methionine aminopeptidases. Biochim Biophys Acta 1477:
157-167.
- Lowther WT, Zhang Y, Sampson PB, Honek JF, Matthews BW (1999).
Insights into the mechanism of
Escherichia coli
methionine
aminopeptidase from the structural analysis of reaction
products and phosphorus-based transition-state analogues.
Biochem 38: 14810-14819.
- Macindoe G, Mavridis L, Venkatraman V, Devignes MD, Ritchie
DW (2010). HexServer: an FFT-based protein-docking
server powered by graphics processors. Nucleic Acid Res
38:
W445-W449.
- Mann M, Jensen ON (2003). Proteomic analysis of post-translational
modifications. Nat Biotechnol 2: 255-261.
- Miljic D, Miljic P, Doknic M, Pekic S, Stojanovic M, Cvijovic G,
Micic D, Popovic V (2013). Growth hormone replacement
normalizes impaired fibrinolysis: new insights into endothelial
dysfunction in patients with hypopituitarism and growth
hormone deficiency, Growth Horm IGF Res 23: 243-248.
- Mitra S, Dygas-Holz AM, Jiracek J, Zertova M, Zakova L,
Holz RC (2006). A new colorimetric assay for methionyl
aminopeptidases: examination of the binding of a new class of
pseudo peptide analogue inhibitors. Anal Biochem 357: 43-49.
- Mukovozov I, Sabljic T, Hortelano G, Ofosu FA (2008). Factors
that contribute to the immmunogenicity of therapeutic
recombinant human proteins. Thromb Haemost 100: 874-882.
- Nudelman I, Akabayov SR, Schnur E, Biron Z, Levy R, Xu Y, Anglister
J (2010). Intermolecular interactions in a 44 kDa interferon -
receptor complex detected by asymmetric reverse-protonation
and two-dimensional NOESY. Biochem 49: 5117-5133.
- Rider P, Carmi Y, Cohen I (2016). Biologics for targeting inflammatory
cytokines, clinical uses, and limitations. Int J Cell Biol
Article
ID 9259646: 11.
- Seal BS (2013). Characterization of bacteriophages virulent for
Clostridium perfringens
and identification of phage lytic
enzymes as alternatives to antibiotics for potential control of
the bacterium. Poult Sci 92: 526-533.
- Shepelkova G, Evstifeev V, Majorov K, Bocharova I, Apt A (2016).
Therapeutic effect of recombinant mutated interleukin 11 in
the mouse model of tuberculosis. J Infect Dis 214: 496-501.
Tripathi NK (2016). Production and purification of recombinant
proteins from
Escherichia coli
. Chem BioEng Rev 3: 116-133.
- Urbano F, Acquafredda A, Aceto G, Penza R, Cavallo L (2012).
Unusual pediatric co-morbility: autoimmune thyroiditis and
cortico-resistant nephrotic syndrome in a 6-month-old Italian
patient. Ital J Pediatr 38: 57.
- Vairo F, Netto C, Dorneles A, Mittelstadt S, Wilke M, Doneda D,
Michelin K, Ribeiro CB, Quevedo A, Vieira T et al. (2013).
Enzyme replacement therapy in a patient with Gaucher disease
type III: a paradigmatic case showing severe adverse reactions
started a long time after the beginning of treatment. JIMD Rep
11: 1-6.
- Vasina JA, Baneyx F (1996). Recombinant protein expression at low
temperatures under the transcriptional control of the major
Escherichia coli
cold shock promoter cspA. Appl Environ
Microbiol 62: 1444-1447.
- Venkatachalam CM, Jiang X, Oldfield T, Waldman M (2003).
LigandFit: a novel method for the shape-directed rapid
docking of ligands to protein active sites. J Mol Graph Model
21: 289-307.
- Wingfield PT (2017). N-terminal methionine processing. Curr
Protoc Protein Sci 88: 6141-6143.
- Xiao Q, Zhang F, Nacev BA, Liu JO, Pei D (2010). Protein N-terminal
processing substrate specificity of
Escherichia coli
and human
methionine aminopeptidases. Biochem 49: 5588-5599.
- Ya LJ, Cui YM, Chen LL, Gu M, Li J, Nan FJ, Ye QZ (2003). Mutations
at the S1 sites of methionine aminopeptidases from
Escherichia
coli
and
Homo sapiens
reveal the residues critical for substrate
specificity. Biochem Biophys Res Comm 307: 172.
- Yang J, Zhang Y (2015). I-TASSER server: new development for
protein structure and function predictions. Nucleic Acid Res
43: W174-W181.
- Zhou Y, Guo XC, Yi T, Yoshimoto T, Pei D (2000). Two continuous
spectrophotometric assays for methionine aminopeptidase.
Analytic Biochem 280: 159-65.