Cloning, Expression, and Characterization of Siamese Crocodile (Crocodylus siamensis) Hemoglobin from Escherichia coli and Pichia pastoris.
Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC-MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV-Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein-ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins.
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand.|Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
Preeyanan
P
Anwised
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand.|Office of the Dean, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
Nisachon
N
Jangpromma
Sriracha Moda Farm, Chon Buri, 20110, Thailand.
Theeranan
T
Temsiripong
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand.|Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
Rina
R
Patramanon
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand.|Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand.
Sakda
S
Daduang
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
Sarawut
S
Jitrapakdee
Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, 869-1404, Japan.
Tomohiro
T
Araki
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand. somkly@kku.ac.th.|Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand. somkly@kku.ac.th.
Sompong
S
Klaynongsruang
D000489:Alligators and Crocodiles; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006454:Hemoglobins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D006454:Hemoglobins; D008024:Ligands; D021122:Protein Subunits; D011994:Recombinant Proteins; D006418:Heme
Crocodile; Escherichia coli; Hemoglobin; Methanol induction; Pichia pastoris; Protein expression