KR100468221B1 - Calcium biosensor to monitor calcium concentration in a cell - Google Patents

Abstract

The present invention stably fluoresces in living cells by inserting a calmodulin gene that binds calcium to the Peridot gene, which is an insertion fluorescent protein into which external genes can be introduced. It relates to a biosensor molecule BCC (bio-cart for calcium) that can measure the amount of calcium in living cells by changing the intensity of fluorescent light according to the amount. In the present invention, the BCC can quantitatively show the presence or absence of a signal causing a change in intracellular calcium by showing a dynamic change in fluorescence intensity according to the intracellular calcium change.

Description

Calcium biosensor to monitor calcium concentration in a cell

Green fluorescence protein (hereinafter referred to as GFP) has been identified in bioluminescent jellyfish. Since GFP is composed of 238 amino acids and does not require other proteins or substrates for fluorescence activity, it is widely used as a reporter for monitoring gene expression and intracellular location of expressed proteins. Has been used in. Various GFP mutations reported to date include enhanced GFP (EGFP) produced by increasing GFP fluorescence, blue fluorescence protein (BFP), cyan fluorescence protein (CFP), and yellow fluorescence protein (YFP) produced by modifying the fluorescence spectrum. . Recently in 1999, Bais et al., Tsien Group, developed a fluorescent GFP mutation even when the GGTGEL amino acid sequence was inserted at the 145 amino acid tyrosine site of the YFP protein, and a calmodulin gene was inserted into the gene. A sensor (calcium sensor) was made and named camgaroo. The calcium sensor developed a new concept of calcium sensor by showing that the fluorescence intensity of YFP changes according to the degree of binding to intracellular calcium. However, this camgaroo shows no fluorescence at 37 ° C, but only at 28 ° C. It was fluorescence and the intensity of fluorescence was low, and had many limitations to be used as a biosensor at the level of tracking calcium concentration in a single cell. In 2001, the same Tsien group was mutated from this Camgaroo, and the strains with fluorescence observed at 37 ° C were selected and named Camgaroo2. Kamgaru 2 was a variant that was able to observe fluorescence at 37 ° C by having a Q69M mutation in which the 69th amino acid glutamine (glutamine) was substituted with methionine. However, Camgaroo2 was also inadequate for measuring calcium at the single cell level, as it was still insignificant at 37 ° C. Therefore, in order to make a calcium biosensor capable of measuring calcium in a single cell, a biosensor showing stable and strong fluorescence at 37 ° C was required.

In the patent application 10-2002-0012409 filed by Neurogenex Co., Ltd., the YGGSGAS amino acid sequence was inserted at amino acid position 145 of the Q69M variant fluorescent protein, and the 192th amino acid proline was changed to leucine. A new fluorescent protein was developed and reported under the name Peridot. Peridot is stable at 37 ° C and 20 times stronger than the previously reported implanted fluorescent proteins, making it a suitable fluorescent protein for making biosensors capable of measuring calcium at the single cell level. Therefore, the present invention has attempted to develop a calcium biosensor capable of measuring calcium concentration in cells in a single cell by introducing a calmodulin gene into the gene.

The present invention relates to the development of a calcium biosensor (calcium biosensor) that can measure the calcium concentration in a single cell for this purpose was to solve the following problems. The first should show stable fluorescence at 37 ° C, and the second should maintain fluorescence intensity enough to track the concentration of calcium in single cells. In addition, the prepared calcium sensor should be sensitive to intracellular calcium concentrations to represent the intracellular calcium concentration through a change in fluorescence.

Figure 1a is a 5 seconds HeLa cell line introduced with BCC fluorescence for 5 seconds while treating (cacacol, 1M calcium ionopore, 100 mM calcium solution, calcium-free solution, etc.) Photo of cells measured in units

FIG. 1B is a graph quantifying the image of FIG. 1A under confocal microscopy

FIG. 1C is a graph in which the graph of FIG. 1B is normalized to fluorescent light before stimulation

In the present invention, in order to achieve the above object, BamHI, NheI restriction enzyme recognition site in the base sequence of peridot (calmodulin) by introducing a calmodulin (calmodulin) gene that can bind to the cell in response to intracellular calcium To develop a calcium biosensor.

Hereinafter, the present invention will be described in detail.

The present inventors used a polymerase chain reaction (PCR) using calmodulin cDNA as a template and using BamHI / CaM F primer 5'-GGGGGATCCATGCATGACCAACTGACAGAA-3 'and NheI / CaM R primer 5'-GGGGCTAGCCTTTGCTGTCATCATTTGTAC-3'. Was performed.

Thereafter, BamHI and NheI restriction enzymes were cloned into the BamHI and NheI restriction enzyme recognition sites in the peridot gene, and the resulting cDNA sequences were analyzed and the recombinant gene was named BCC (bio-cart for calcium). The prepared BCC was introduced into the HeLa cell line and incubated at 37 ° C. for at least 24 hours. Since the BCC-introduced cells were placed under a confocal microscope, fluorescence was confirmed while irradiating 488 nm light using an argon laser. In the BCC, the calmodulin domain is located in the loop portion of the cylinder-shaped YFP, and the structural change of BCC induced by the degree of calmodulin-calcium binding is thought to be a change in fluorescence.

By measuring the change in intracellular calcium using HeLa cells into which BCC was introduced, the invention could be completed by confirming the fluorescence change of BCC according to the change of intracellular calcium concentration. HeLa cells were introduced with BCC while sequentially treating 10M cabacol, 1M calcium ionopore, 100 mM calcium solution, calcium-free solution, etc. The fluorescence of the cells was measured. Figure 1a is an array of images measured at intervals of 5 seconds, Figure 1b is a graph showing the change in intracellular calcium with time for the four cells shown in Figure 1a. According to the degree of introduction of the BCC gene, the four cells show different degrees of fluorescence, but they can be seen to respond in the same manner by external stimulation. This can be clearly seen by normalizing the fluorescent light before stimulation as shown in FIG. 1C.

From the above results, it can be seen that the BCC responds sensitively to external stimuli, and the increase in fluorescence induced by the same stimulus is maintained for a certain period of time. In the high-speed evaluation system that is widely used now, considering that the time required to measure the first well to the last well is 96 minutes for the 96 well type, the BCC that maintains the fluorescent light for a certain period (5 minutes) for the same stimulus is The existing calcium indicator is considered to be a calcium biosensor that can be applied to various experiments that cannot be measured under high-speed evaluation system.

Bio-cart for Calcium (BCC) according to the present invention is a fluorescent protein incorporating the calmodulin gene into the paridot, a type of inserted fluorescent protein (Inserted YFP), which shows a stable and strong fluorescence at 37 ° C, and the intracellular calcium concentration. The intensity of the fluorescence changes according to the change of calcium biosensor (calcium biosensor). In addition, the BCC is considered to be useful for a high-speed evaluation system for a large number of samples because the fluorescent light increased by a single stimulus is kept constant for more than 5 minutes.

Although the present application is not shown as an example, it is possible to prepare a cell-level calcium analysis system and a transgenic disease model animal using the same by selecting a cell line into which the BCC of the present invention can be used for drug development. It is obvious to me.

<110> NEUROGENEX CO. LTD. <120> Calcium biosensor to monitor calcium concentration in a cell <160> 2 <170> Kopatent In 1.71 <210> 1 <211> 1182 <212> DNA <213> Aequorea victoria <220> <221> CDS <222> (1 (1179) <223> Calmodulin Inserted peridot <400> 1 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg act acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tac ggc ctg atg tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag c tg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac ggt gga tcc atg cat gac caa ctg aca gaa gag cag atc gca 480 Asn Tyr Gly Gly Ser Met His Asp Gln Leu Thr Glu Glu Gln Ile Ala 145 150 155 160 gaa ttt aaa gag gct ttc tcc cta ttt gac aag gac ggg gat ggg aca 528 Glu Phe Lys Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Thr 165 170 175 ata aca acc aag gag ctg ggg acg gtg atg cgg tct ctg ggg cag aac 576 Ile Thr Thr Lys Glu Leu Gly Thr Val Met Arg Ser Leu Gly Gln Asn 180 185 190 ccc aca gaa gca gag ctg cag gac atg atc aat gaa gta gat gcc gac 624 Pro Thr Glu Ala Glu Leu Gln Asp Met Ile Asn Glu Val Asp Ala Asp 195 200 205 ggt aat ggc aca atc gac ttc cct gag ttc ctg aca atg atg gca aga 672 Gly Asn Gly Thr Ile Asp Phe Pro Glu Phe Leu Thr Met Met Ala Arg 210 215 220 aaa atg aaa gac aca gac agt gaa gaa gaa att aga gaa gcg ttc cgt 720 Lys Met Lys Asp Thr Asp Ser Glu Glu Glu Ile Arg Glu Ala Phe Arg 225 230 235 240 gtg ttt gat aag gat ggc aat ggc tac atc agt gca gca gag ctt cgc 768 Val Phe Asp Lys Asp Gly Asn Gly Tyr Ile Ser Ala Ala Glu Leu Arg 245 250 255 cac gtg atg aca aac ctt gga gag aag tta aca gat gaa gag gtt gat 816 His Val Met Thr Asn Leu Gly Glu Lys Leu Thr Asp Glu Glu Val Asp 260 265 270 gaa atg atc agg gaa gca gac atc gat ggg gat ggt cag gta aac tac 864 Glu Met Ile Arg Glu Ala Asp Ile Asp Gly Asp Gly Gln Val Asn Tyr 27 5 280 285 gaa gag ttt gta caa atg atg aca gca aag gct agc aac agc cac aac 912 Glu Glu Phe Val Gln Met Met Thr Ala Lys Ala Ser Asn Ser His Asn 290 295 300 gtc tat atc atg gcc gac aag cag aag aac ggc atc aag gtg aac ttc 960 Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe 305 310 315 320 aag atc cgc cac ac atc gag gac ggc agc gtg cag ctc gcc gac cac 1008 Lys Ile Arg His Asn Asp Gly Ser Val Gln Leu Ala Asp His 325 330 335 tac cag cag aac acc ccc atc ggc gac ggc ctc gtg ctg ctg ccc gac 1056 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp 340 345 350 aac cac tac ctg agc tac cag tcc gcc ctg agc aaa gac ccc aac gag 1104 Asn His Tyr Leu Ser Ty r Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 355 360 365 aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc 1152 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 370 375 380 act atc ggc atg gac gag ctg tac aag t aa 1182 Thr Ile Gly Met Asp Glu Leu Tyr Lys 385 390 <210> 2 <211> 393 <212> PRT <213> Aequorea victoria <400> 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Tr p Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Gly Gly Ser Met His Asp Gln Leu Thr Glu Glu Gln Ile Ala 145 150 155 160 Glu Phe Lys Glu Ala Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Thr 165 170 175 Ile Thr Thr Lys Glu Leu Gly Thr Val Met Arg Ser Leu Gly Gln Asn 180 185 190 Pro Thr Glu Ala Glu Leu Gln Asp Met Ile Asn Glu Val Asp Ala Asp 195 200 205 Gly Asn Gly Thr Ile Asp Phe Pro Glu Phe Leu Thr Met Met Ala Arg 210 215 220 Lys Met Lys Asp Thr Asp Ser Glu Glu Glu Ile Arg Glu Ala Phe Arg 225 230 235 240 Val Phe Asp Lys Asp Gly Asn Gly Tyr Ile Ser Ala Ala Glu Leu Arg 245 250 255 His Val Met Thr Asn Leu Gly Glu Lys Leu Thr Asp Glu Glu Val Asp 260 265 270 Glu Met Ile Arg Glu Ala Asp Ile Asp Gly Asp Gly Gln Val Asn Tyr 275 280 285 Glu Glu Phe Val Gln Met Met Thr Ala Lys Ala Ser Asn Ser His Asn 290 295 300 Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe 305 310 315 320 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His 325 330 335 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp 340 345 350 Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 355 360 365 Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile 370 375 380 Thr Ile Gly Met Asp Glu Leu Tyr Lys 385 390

Claims (3)

A recombinant fluorescent protein for measuring intracellular calcium concentration, having the amino acid sequence of SEQ ID NO: 2. The gene base sequence of SEQ ID NO: 1 encoding the recombinant fluorescent protein of claim 1. A method of measuring intracellular calcium concentration using the recombinant fluorescent protein of claim 1 or the nucleotide sequence of claim 2.