Jurnal ILMU DASAR, Vol. 24 No. 2, Juli 2023 : 151-158 151 Journal homepage: https://jurnal.unej.ac.id/index.php/JID Kontruksi dan Ekspresi Protein Rekombinan Wee1 pada Escherichia coli strain BL21 (DE3) Construction and expression of Wee1 recombinant protein in Escherichia coli strain BL21 (DE3) Ladefa Primana Oktapan 1 , Ardela Alief Maulani 1 , Netty Ermawati 2 , Bambang Sugiharto 3,4*) 1 Program Studi Agroteknologi, Fakultas Pertanian, Universitas Jember 2 Jurusan Produksi Pertanian, Politeknik Negeri Jember 3 Jurusan Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Jember 4 UPT Laboratorium Terpadu dan Sentra Inovasi Teknologi CDAST, Universitas Jember *E-mail: sugiharto.fmipa@unej.ac.id ABSTRACT Wee1 is a gene encoding for protein kinase that is located in the nucleus and it plays an essential role in determining the timing of mitosis. Overexpression of Wee1 in rice is resulting in increased plant size. However, the increasing plant size due to the increase of Wee1 protein expression is not elucidated. Immunodiagnostic using specific antibodies against Wee1 protein should be conducted to determine the role of the protein on cell division. This experiment was directed to construct Wee1 in an expression vector for synthesis of recombinant Wee1 protein using three strategies of construction. The three strategies were construction of full length (FL), deletion of putative binding site (BS), and deletion of N-terminal domain (ΔN) of Wee1. Restriction analysis with BamH1/Sal1 of FL-Wee1, BS-Wee1, and ΔN-Wee1 construct resulted in the different DNA fragments with molecular size at 1239, 1176, and 960bp, respectively. This result indicated that the Wee1 fragments have been successfully inserted in the expression plasmid which was further confirmed using DNA sequencing. Colony PCR analysis showed that Escherichia coli strain BL21 (DE3) has been transformed with the constructs. The protein analysis using SDS-PAGE revealed that the recombinant protein of Wee1 synthesized in the E.coli containing ΔN-Wee1 construct, but not in FL-Wee1 and BS-Wee1 constructs. The ΔN-Wee1 protein was synthesized in an insoluble fraction with a molecular size of 38.8 kDa which is the same as the size estimated using the software ExPASy. Interestingly, the level of synthesized ΔN-Wee1 protein was not induced by IPTG concentration. Collectively, the results indicated that the DNA construct of ΔN-Wee1 is suitable for recombinant protein production. Keywords: Weel, construction, synthesis, recombinant protein, Escherichia coli. PENDAHULUAN Wee1 merupakan protein kinase yang berperan penting sebagai negatif regulator pada regulasi pembelahan sel ((Ko et al., 2018; Nurse & Thuriaux, 1980). Protein Wee1 berpengaruh pada ukuran sel dengan menghambat regulasi pembelahan sel pada saat check point fase mitosis. Peran Wee1 dipengaruhi oleh regulasi post-translasi yaitu protein Wee1 terfosforilasi untuk menjalankan fungsinya (Qiu et al., 2017). Saat proses pembelahan sel, protein Wee1 memphosphorilasi Cyclin-dependent kinase (CDKs) dalam kompleks CDKs-Cyclin B dan berdampak pada penghambatan proses pembelahan sel (Lucena et al., 2017). Protein Wee1 dilaporkan mempunyai peranan penting dalam perkembangan biji jagung (Sun et al., 1999). Overekspresi gen pengkode Wee1 diketahui menyebabkan peningkatan ukuran sel meristem akar pada Arabidopsis (Siciliano et al., 2019) dan meningkatkan ekspansi sel selama proses perkembangan buah pada tanaman tomat (Gonzalez et al., 2007). Pada tanaman padi dihipotesiskan bahwa overeskpresi gen Wee1 dapat meningkatkan produksi, sehingga dilakukan isolasi gen Wee1 (Ermawati & Wibisono, 2017). Lebih lanjut, over ekspresi gen Wee1 menghasilkan tanaman padi transgenik dengan morfologi daun berwarna hijau pekat dan ukuran tanaman lebih besar dari pada tanaman padi non-transgenik (Prasetyo et al., 2018) Untuk mengetahui analisis dan regulasi protein Wee1 diperlukan analisa pada tingkat protein Wee1. Analisis molekuler dapat dilakukan pada tingkat transkripsi atau ekspresi gen, maupun pada tingkat translasi atau proteinnya. Analisis biokimia tingkat protein perlu dilakukan untuk menentukan hubungan antara perubahan morfologi fenotipik dengan