Mehmet Inan | Akdeniz University (original) (raw)

Papers by Mehmet Inan

Methods in Molecular Biology, 2019

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is used as an expression system f... more The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is used as an expression system for recombinant protein production for a variety of applications. It grows rapidly on inexpensive media containing methanol, glucose, glycerol, or ethanol as a sole carbon source. P. pastoris makes many posttranslational modifications and produces recombinant proteins either intracellularly or extracellularly. Because of these properties, P. pastoris has become a highly preferred host organism for biotechnology, pharmaceutical industry, and researchers.Recombinant protein production is usually performed under the control of the promoter of the alcohol oxidase gene I (AOX1). The AOX1 promoter is induced by methanol and repressed by glucose and ethanol. The regulation mechanisms of the AOX1 promoter have been studied in recent years. Another promoter used in recombinant protein production is derived from glyceraldehyde 3-phosphate dehydrogenase (GAP). It is a constitutive promoter. Recent literature showed that newly identified promoters of P. pastoris are promising as well, in addition to pAOX1 and pGAP.In this chapter, the regulation mechanisms of inducible pAOX1 and constitutive pGAP promoters are discussed. In addition, here we present an overview about the novel ADH3 promoter and alternative promoters of P. pastoris.

Trakya University Journal of Natural Sciences, 2018

In this study, it was aimed to optimise the extracellular peptidase production of Bacillus amylol... more In this study, it was aimed to optimise the extracellular peptidase production of Bacillus amyloliquefaciens FE-K1, previously isolated from ropy wholemeal bread, by using response surface methodology (RSM) based on central composite design (CCD). The temperature (20-45°C), initial pH of the enzyme production medium (pH 5-9) and inoculation level (1-5%, v/v) were used as the factors for RSM, and the fermentation time was determined for each trial separately. Results showed that the optimum peptidase production occurred at 33.4°C, pH 6.62 and 2.3% inoculation. It was determined that the fermentation time was only 7h, the crude enzyme had a peptidase activity of 49.17U/mL and a specific activity of 504.77U/mg under the optimised conditions.

Yeast, 2019

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely ... more The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviors with ADH900 on glucose, glycerol and methanol, however, it is strongly induced by ethanol.

Protein Expression and Purification, 2019

In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutan... more In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutant strain derived from B. subtilis 168) were transformed into P. pastoris KM71H. Extracellular recombinant protein production was achieved with methanol induction under the regulation of AOX1 promoter utilizing the Saccharomyces cerevisiae α-mating factor sequence for extracellular secretion. The molecular weight of the recombinant enzymes BK07pul and PY22pul were both approximately 90 kDa. Both enzymes showed highest activity at 40°C, however PY22pul showed optimum activity at pH 6 whereas, BK07pul had highest activity at pH8. BK07pul and PY22pul activities were determined as 8.46 U/mL and 15 U/mL. The enzyme stability of BK07pul was higher (89%) than PY22pul (68%) where relative activity was determined as activity remaining after 1 hour at corresponding optimum conditions for each. Amino acid homology evaluation revealed the two enzymes had 80% identity in primary structure. The presence of conserved sequences consisting of 7 amino acids (YNWGYDP) in both enzymes confirmed these to be type I pullulanases, capable of hydrolyzing α-1,6 glucosidic bonds of pullulan resulting in maltotriose units.

Pichia Protocols, Second Edition

Abstract Human hookworm infection is one of the most significant parasitic infections, and a lead... more Abstract Human hookworm infection is one of the most significant parasitic infections, and a leading global cause of anemia and malnutrition of adults and children in rural areas of the tropics and subtropics. Necator americanus secretory protein (Na-ASP1), which is a potential vaccine ...

Methods in molecular biology (Clifton, N.J.), 2007

The N-terminal and C-terminal portions of the heavy chain fragment C from botulinum neurotoxin se... more The N-terminal and C-terminal portions of the heavy chain fragment C from botulinum neurotoxin serotype C [rBoNT(HC)] were expressed in Pichia pastoris and purified by ion-exchange chromotography (IEC). The N-terminal fragment, rBoNTC(Hc)-N, was purified in three IEC steps: a Q Sepharose Fast Flow (FF) capture step followed by a negative SP Sepharose FF step, and finally, Q Sepharose FF as a polishing step. The purification process resulted in greater than 90% pure rBoNTC(Hc)-N based on SDS-PAGE, and yielded up to 1.02 g of rBoNTC(Hc)-N/kg of cells. Alternately, the C-terminal fragment, rBoNTC(Hc)-C, was purified by using a SP Sepharose FF capture step followed by a second SP Sepharose FF step, and finally a Q Sepharose FF as a polishing step. This purification process resulted in greater than 95% pure rBoNTC(Hc)-C based on SDS-PAGE, and yielded up to 0.2 g of rBoNTC(Hc)-C/kg cells. The final protein yield is a function of protein expression level during fermentation and the purific...

Biotechnology Progress, 2011

in Wiley Online Library (wileyonlinelibrary.com). Ricin is a potent toxin and a potential bioterr... more in Wiley Online Library (wileyonlinelibrary.com). Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A-chain (RTA), which is an N-glycosidase enzyme, and the B-chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1-33/ 44-198 protein, herein denoted RVEa TM) expressed in Escherichia coli using a codon-optimized gene was shown to be non-toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale-up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)-compliant production of RVEc TM at the 40 L scale. The average yield of the final purified bulk RVEc TM is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc TM was [99% pure by three HPLC methods and SDS-PAGE. The intact mass and peptide mapping analysis of RVEc TM confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc TM was immunoprotective against lethal ricin challenge and elicited neutralizing anti-ricin antibodies in 95-100% of the vaccinated mice.

ACS Nano, 2010

Process to make the nanoparticle necklace network in solution: Au nanoparticles 10 and 60 nm in d... more Process to make the nanoparticle necklace network in solution: Au nanoparticles 10 and 60 nm in diameter with concentrations 5.7 x 10 12 and 2.6 x 10 10 particles mL-1 (pH 6 and 8), respectively, are purchased from BBI International. The particles are electrostatically stabilized with a negative charge due to citrate capping. An aqueous suspension of 60 and 10 nm particles at a volume ratio of 1:6, respectively, is prepared. The network is fabricated by adding 120 µL of a 10 mM HCl solution drop-wise to 270 µL of the Au particle suspension under constant stirring. After the complete addition of HCl, the solution turns from wine-red to violet-blue in approximately 18 hours. The blue color is indicative of necklace formation (see Fig. 1 in MS). Once the necklaces have formed, the solution remains stable for at least one week. The pH of the blue solution is ~2.3. Characterization tools: The FESEM is a Hitachi S-4700 Field-Emission Scanning Electron Microscope; the UV-vis spectrometer is Ocean Optics Model USB2000, and I-V measurement is conducted using a home-built system using a high sensitivity multimeter (Agilent 3458A) to measure current.

Journal of Molecular Catalysis B: Enzymatic, 2010

... Amplification of the amyE gene was performed according to [14] using a TGradient (Whatman-Bio... more ... Amplification of the amyE gene was performed according to [14] using a TGradient (Whatman-Biometra) thermocycler with the primers AmyFPmlI (5′-TCA CGT GGA TGT TTG CAA AAC GAT TCA AAA-3′) and AmyRKpn (5′-AGG TAC CTC AAT GGG GAA GAG AAC C-3 ...