Preparation of Recombinant Protamine and Analysis of Its Antibacterial Activity

doi:10.13560/j.cnki.biotech.bull.1985.2021-0076

Abstract

Abstract:

This work aims to express recombinant protamine by prokaryotic expression system and to detect its biological activity. First,genetic engineering was used to obtain CYGBm by mutating all the methionine ATG sites（except the start codon）into ATC. Then,CYGBm and protamine fusion method was applied to express the recombinant protamine. CNBr was employed to cleave the methionine sites in the CYGBm and protamine and thus it was purified. Finally,tube culture method was to analyze the antimicrobial activity of the recombinant protamine and its application effect was detected by fresh shrimp. As results,the fusion of the CYGBm and the protamine could be expressed in a large number of inclusion bodies in the prokaryotic expression system. The purified recombinant protamine was prepared by separation and CNBr lysis. It was verified that the recombinant protamine demonstrated excellent antibacterial activity. In conclusion,the preparation of recombinant protamine with antibacterial activity can be achieved by genetic engineering method,which provides a theoretical basis for industrial production of recombinant protamine.

Key words: protamine, mutation, fusion, bacteriostatic

LI Zhao-fa, QIU Dan-dan. Preparation of Recombinant Protamine and Analysis of Its Antibacterial Activity[J]. Biotechnology Bulletin, 2021, 37(10): 162-168.

Figures/Tables 7

Table 1 Oligonucleotide sequences involved in this study

Oligonucleotide name	Sequence（5'-3'）
SEQ ID NO.1	AACATATGGAGAAAGTGCCAGGCGAG
SEQ ID NO.2	GGCCCAGATAGCCTGCACCGCCTTCC
SEQ ID NO.3	CGGTGCAGGCTATCTGGGCCCGGCTC
SEQ ID NO.4	CTCCAGGGGATCTTCGATGTGCTTGAACTGGC
SEQ ID NO.5	CACATCGAAGATCCCCTGGAGATCGAGCGGAGC
SEQ ID NO.6	GAGGGCCCCGATGACTCGGCAGGCGTGC
SEQ ID NO.7	GCCGAGTCATCGGGGCCCTCAACAC
SEQ ID NO.8	CAGGATCCCGGCCCCGAAGAGGGCAGTG

Fig. 1 Flow chart of fixed point mutations CYGB by overlapping extended PCR

Fig. 2 Whole strains SDS-PAGE of CYGB and CYGBm M:Marker. 1:CYGB. 2:CYGBm

Fig. 3 Nucleic acid electrophoresis of recombinant plasmid pET22 B-CYGBm-Pro by PCR and double enzyme digestions M1:15 000 bp marker DNA. 1:PCR of BL21 bacterial colony. 2:PCR of BL21-pET22b-CYGBm-Pro bacterial colony. 3:Plasmid of pET22b-CYGBm-Pro by double enzyme digestions of Nde I/Eco I. M2:2 000 bp marker DNA

Fig. 4 SDS-PAGE of CYGBm-Pro expression and purified CYGBm-Pro and rPro M1:marker. 1:Uninduced whole bacterium. 2:Induced whole bacterium. 3:Supernatant of induced whole bacterium. 4:Sediment of induced whole bacterium. 5:Purified CYGB-Pro. 6:Purified rPro

Fig. 5 Curves of growth inhibition rates of different bacteria with different concentrations of rPro

Fig. 6 Curves of pH values（A）and TVB-N values（B）of shrimp treated by rPro during storage

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