Molecular characterization and tissue expression of carboxypeptidase H (CPH) gene in flounder (Paralichthys olivaceus)

Abstract

Carboxypeptidase H (CPH) is one of several enzymes required for the processing of peptide hormone precursors and removal of basic amino acids from intermediates during protein biosynthesis. The CPH gene was isolated from a flounder (Paralichthys olivaceus) brain cDNA library and was found to consist of 1,590 bp encoding 455 amino acids residues. The nucleotide sequence of the CPH gene was compared with those of other species, including zebrafish and mouse, and turned out to be conserved during evolution. Expression of CPH mRNA was confirmed by both Reverse transcription polymerase Chain Reaction (RT-PCR) and Northern blot analysis. A recombinant protein of approximately 50 kDa, which corresponds to CPH, was functionally expressed in E. coli. Biochemical studies on the expressed protein were performed. We report here the molecular characteristics and tissue expression of newly identified CPH cDNA from flounder (P. olivaceus).

Molecular Cloning and Co-Expression of Phytoene Synthase Gene from Kocuria gwangalliensis in Escherichia coli

“…The proteins were electrotransferred from the SDS-PAGE gel to a nitrocellulose membrane, probed with goat antiserum against 6-His tag, and incubated with alkaline phosphatase coupled to goat antibody against goat IgG. The nitrocellulose membrane was developed using BCIP/NBT (Sigma-Aldrich, USA) [27]. Separately, the crtE and crtI genes from P. haeundaensis were inserted into the pRSFDuet-1 vector (Novagen, USA) to synthesize lycopene in E. coli BL21 (DE3).…”

Cloning of Geranylgeranyl Pyrophosphate Synthase (CrtE) Gene from Kocuria gwangalliensis and Its Functional Co-expression in Escherichia coli

“…대장균 발현 벡터의 구축 및 라이코펜 생합성을 위한 재조 합 DNA 구축 확보된 crtE 유전자를 대장균에서 과발현시키기 위해서 PAGE gel에 의해 분리된 C-말단 부위에 6X-histidine tag을 가진 재조합 단백질을 electro-transfer을 이용하여 nitrocellulose (NC) membrane로 이동 시 켰으며, 이들 재조합 단백질의 확인을 위해 1차 항체는 goat antiserum against 6-His tag를 사용하였으며, 2차 항체는 alkaline phosphate가 결합된 goat antibody against goat IgG 항체를 사용하였다. 또한, 이들 항체의 결합에 의한 NC membrane 상에서의 발색을 위한 시약은 BCIP/NBT (Sigma-Aldrich, St. Louis, MO, USA) 시약을 사용하여 대장균 내 재 조합 단백질인 KgCrtE의 발현 유무를 확인하였다[18]. 또한, 대장균 BL21 (DE3)에서 라이코펜(lycopene) 생합성을 위해 P.…”